Nucleotide sequences encoding cry1bb proteins for enhanced expression in plants

ABSTRACT

The present invention describes compositions and methods that are useful in the control of lepidopteran insect pests, and more particularly describes nucleotide sequences for use in plants that encode full-length and truncated insecticidal toxins, as well as chimeric toxins. The nucleotide sequences of the present invention exhibit modifications that, when compared to the native sequences obtained from  Bacillus thuringiensis  species, make them particularly useful for enhanced, improved, and or optimized expression in monocot and dicot plant species. Using methods well known to those skilled in the art the nucleotide sequences described herein can be used to transform plant cells and plant tissue in order to produce transgenic plants that express the encoded proteins, therefore conferring upon the transgenic plants the ability to resist insect infestation.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to transgenic plants exhibitinginsecticidal activity, and to DNA constructs containing genes encodingCry1Bb proteins for conferring insect resistance when expressed inplants. More specifically, the present invention relates to a method ofexpressing at least one insecticidal protein in a plant transformed witha gene encoding an insecticidal fragment of a B. thuringiensisδ-endotoxin, resulting in effective control of susceptible target pests.

1.2 Description of Related Art

1.2.1 Methods of Controlling Insect Infestation in Plants TheGram-positive soil bacterium B. thuringiensis is well known for itsproduction of proteinaceous parasporal crystals, or δ-endotoxins, thatare toxic to a variety of lepidopteran, Coleopteran, and Dipteranlarvae. During the sporulation phase of growth, B. thuringiensisproduces crystal proteins that are each specifically toxic to certainspecies of insects. Many different strains of B. thuringiensis have beenshown to produce insecticidal crystal proteins. Compositions comprisingB. thuringiensis strains that produce proteins exhibiting insecticidalactivity have been used commercially as environmentally acceptabletopical insecticides because of their toxicity to the specific targetinsect pests, and non-toxicity to plants and other non-targetedorganisms.

δ-endotoxin crystals are toxic to insect larvae upon ingestion of thecrystalline protein composition. Solubilization of the crystal in thealkaline midgut of the insect releases the protoxin form of theδ-endotoxin that, in most instances and particularly for Cry1 typetoxins, is subsequently processed to an active toxin by one or moremidgut proteases. The activated toxins recognize and bind to thebrush-border of the insect midgut epithelium through receptor proteins.Several putative crystal protein receptors have been isolated fromcertain insect larvae (Knight et al. 1994, Mol. Microbiol. 11:429-436;Gill et al. 1995, Molecular action of insecticides on ion channels, pp.308-319, Clark, J. M. Editor; Masson et al. 1995, J. Biol. Chem.270:11887-11896). The binding of active toxins is followed byintercalation and aggregation of toxin molecules to form pores withinthe midgut epithelium. This process leads to osmotic imbalance,swelling, lysis of the cells lining the midgut epithelium, and eventuallarvae mortality.

1.2.2 Transgenic B. thuringiensis δ-Endotoxins as Biopesticides

Plant resistance and biological control are central tactics of controlin the majority of insecticide improvement programs applied to the mostdiverse crops. With the advent of molecular genetic techniques, variousδ-endotoxin genes have been isolated and their DNA sequences determined.These genes have been used to construct certain genetically engineeredB. thuringiensis products that have been approved for commercial use.Recent developments have seen new &endotoxin delivery systems developed,including plants that contain and express genetically engineeredδ-endotoxin genes. Expression of B. thuringiensis δ-endotoxins in plantsholds the potential for effective management of plant pests so long ascertain problems can be overcome. These problems include the developmentof insect resistance to the particular Cry protein expressed in theplant, expression in the same plant of two or more insecticidally activeproteins toxic to the same insect species and each exhibiting differentmodes of action, and the presence of the transgene or other elementswithin the expression cassette in which the transgene resides causingcommercially unacceptable morphologies in the transgenic selectedevents.

Expression of B. thuringiensis δ-endotoxins in transgenic cotton, corn,and potatoes has proven to be an effective means of controllingagriculturally important insect pests (Perlak et al. 1990, BioTechnology8:939-943; Perlak et al. 1993, Plant Mol. Biol. 22:313-321). Transgeniccrops expressing B. thuringiensis δ-endotoxins enable growers tosignificantly reduce the application of costly, toxic, and sometimesineffective topical chemical insecticides. Use of transgenes encoding B.thuringiensis δ-endotoxins is particularly advantageous when insertionof the transgene has no negative effect on the yield of desired productfrom the transformed plants. Yields from crop plants expressing certainB. thuringiensis δ-endotoxins such as Cry1A or Cry3A have been observedto be equivalent to or better than otherwise similar non-transgeniccommercial plant varieties. This indicates that expression of some B.thuringiensis δ-endotoxins does not have a significant negative impacton plant growth or development. This is not the case, however, for allB. thuringiensis δ-endotoxins that may be used for expression in plants.

The use of topical B. thuringiensis-derived insecticides may also resultin the development of insect strains resistant to the insecticides.Resistance to Cry1A B. thuringiensis δ-endotoxins applied as foliarsprays has evolved in at least one well-documented instance (Shelton etal., 1993, J. Econ. Entomol. 86:697-705). It is expected that insectsmay similarly develop resistance to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of corn, cotton,potato, and other germplasm containing genes encoding B. thuringiensisδ-endotoxins. One possible way to coordinately increase theeffectiveness of the insecticide against target pests and to reduce thedevelopment of insecticide-resistant pests would be to ensure thattransgenic crops express high levels of B. thuringiensis δ-endotoxins(McGaughey and Whalon 1993, Science 258:1451-55; Roush 1994, BioControlSci. Technol. 4:501-516).

In addition to producing a transgenic plant that expresses B.thuringiensis δ-endotoxins at high levels, commercially viable B.thuringiensis genes must satisfy several additional criteria. Forinstance, expression of these genes in transgenic crop plants must notreduce the vigor, viability or fertility of the plants, nor should itaffect the normal plant morphology. Such detrimental effects haveundesired results: they may interfere with the recovery and propagationof transgenic plants; they may also impede the development of matureplants, or confer unacceptable agronomic characteristics.

There remains a need for compositions and methods useful in producingtransgenic plants that express B. thuringiensis δ-endotoxins at levelshigh enough to effectively control target plant insect pests as well asprevent the development of insecticide-resistant pest strains. A methodresulting in higher levels of expression of the B. thuringiensisδ-endotoxins will also provide the advantages of more frequentattainment of commercially viable transformed plant lines and moreeffective protection from infestation for the entire growing season.

There also remains a need for a method of increasing the level of inplanta expression of B. thuringiensis δ-endotoxins that does notsimultaneously result in plant morphological changes that interfere withoptimal growth and development of desired plant tissues. For example,the method of potentiating expression of the B. thuringiensisδ-endotoxins in maize should not result in a corn plant which cannotoptimally develop for cultivation and harvest of the crop.

Additionally, there remains a need for compositions and methods usefulin producing transgenic plants which express two or more Bacillusthuringiensis δ-endotoxins toxic to the same insect species and whichconfers a level of resistance management for delaying the onset ofresistance of any particular susceptible insect species to one or moreof the insecticidal agents expressed within the transgenic plant.Alternatively, expression of a Bacillus thuringiensis insecticidalprotein toxic to a particular target insect pest along with a differentproteinaceous agent toxic to the same insect pest but which conferstoxicity by a means different from that exhibited by the Bacillusthuringiensis toxin is desirable. Such other different proteinaceousagents comprise Xenorhabdus sp. or Photorhabdus sp. insecticidalproteins, deallergenized and de-glycosylated patatin proteins orpermuteins thereof, Bacillus thuringiensis vegetative insecticidalproteins, lectins, and the like. One means for achieving this resultwould be to produce two different transgenic events, each eventexpressing a different insecticidal protein, and breeding the two traitstogether into a hybrid plant. Another means for achieving this resultwould be to produce a single transgenic event expressing bothinsecticidal genes. This can be accomplished by transformation with anucleotide sequence that encodes both insecticide proteins, but anothermeans would be to produce a single event that was transformed to expressa first insecticide gene, and then transform that event to produce aprogeny event that expresses both the first and the second insecticidegenes.

Achievement of these goals such as sufficient co-expression of multipleinsecticidally active proteins in the same plant, and/or high expressionlevels of insecticidal proteins which do not result in aberrantmorphological effects upon the transgenic plant has been elusive, andtheir pursuit has been an ongoing and important aspect of the long termvalue of insecticidal plant products.

More than two-hundred and fifty individual insecticidal proteins havebeen identified from Bacillus thuringiensis species, but only a handfulof these have been tested for expression in plants. Initially, thenative sequences were utilized in plant expression cassettes, and theseproved useless for producing transgenic plants exhibiting insecticidalproperties. This was likely due to the fact that native Bacillusthuringiensis nucleotide sequences exhibit a nucleotide compositionsubstantially different from that in plants. Modifications to sequencesencoding Bacillus thuringiensis toxin proteins which substantiallyreduces the AT nucleotide composition results in substantialimprovements in levels of expression of some of these proteins inplants, however, expression of Bacillus thuringiensis δ-endotoxins inplants is not without effect. It requires trial and errorexperimentation to determine which if any Bacillus thuringiensisδ-endotoxin protein when expressed in planta will produce a commerciallyuseful plant, which exhibits levels of expression that are effective incontrolling target insect pests, and which does not result inmorphologically abnormal effects upon the plant. Examples of Bt proteinsthat have been successfully expressed in plants are substantiallylimited to Cry1Ab, Cry1Ac, Cry2Ab, amino acid sequence variants ofCry3Bb, Cry1C, and Cry3C. Cry2Ab was only successfully expressed whentargeted for importation into chloroplasts. Cry1 proteins have beenexpressed in plants as full-length protoxins exhibiting an amino acidsequence that is substantially similar to the form in which they arefound in nature when expressed by Bacillus thuringiensis species. Cry1proteins have also been expressed in plants as less than full-lengthforms of the protein, comprising essentially the tryptic core or activetoxin domain of the Cry1 protein. However, Cry1 proteins have not beenexpressed at high levels. Since the majority of acreage planted on anannual basis with recombinant plants exhibiting insecticidal bioactivityconsists substantially of plants expressing Cry1A proteins, thelikelihood of the onset of resistance to Cry1A proteins by target insectpest species is greater than it would be if a second mode of action ofinsect control was also packaged in some way or expressed along with thecry1 allele, or if the cry1 allele was expressed at high levels.

To date, no field resistance has been observed. However, there have beenseveral examples of acquired resistance to Cry1A proteins underlaboratory conditions. Therefore, it is imperative that plants currentlyexpressing only one Cry protein be replaced with plants containingadditional genes encoding insecticidal proteins exhibiting differentmechanisms of insecticidal activity. Thus, the discovery of new Bacillusthuringiensis isolates and new uses of known Bacillus thuringiensisisolates remains an empirical and unpredictable art. There also remainsa need for new toxin genes that can be expressed at adequate levels inplants in a manner that will result in the effective control of targetinsect pest species.

2.0 SUMMARY OF THE INVENTION

The present invention provides compositions and methods for use incontrolling target insect pests, and in particular lepidopteran insectpest species susceptible to Cry1Bb insecticidal crystal proteins orinsecticidal variants thereof. More specifically the subject inventionprovides expression cassettes for use in plants, the expressioncassettes containing at least nucleotide sequences encoding the fulllength Cry1Bb protein, or variants thereof, which exhibit at least thelevel of insecticidal activity as the native full length Cry1Bb protein,or insecticidally active fragments thereof, which confer insectinhibitory traits to a plant expressing the protein from within thecassette provided. The nucleotide sequences of the present inventionencoding Cry1B proteins or insecticidal fragments thereof containmodifications in comparison to the native Bacillus thuringiensis cry1Bbcoding sequence which result in improved expression of the Cry1Bbprotein in plants compared to expression levels observed in plants usingthe native Bt cry1Bb coding sequence, and which make these sequencesparticularly well suited for expression of the Cry1Bb protein in plants.

The invention provides in one embodiment nucleotide sequences exhibitingCry1Bb variant coding sequences that are optimized for expression inplants to produce an insect inhibitory amount of a Cry1Bb protein orinsecticidal fragment thereof which is toxic or inhibitory to one ormore target lepidopteran insect pest species. These nucleotide sequencesinclude plant preferred Cry1Bb coding sequences as set forth in SEQ IDNO:3, 5, 8, 11, and 13, or as contained within the vectors or nucleotidesequence fragments corresponding to pMON33733, pMON33734, pMON40227, andpMON40228. Those skilled in the art will recognize that these sequences,in particular the sequences as set forth in the SEQ ID NO's herein, canbe artificially synthesized and introduced into any vector of interestfor use in expressing the sequences disclosed herein or sequencessubstantially the same as those set forth herein in plants. Suchsequences are prepared by extrapolating a preferred nucleotide sequencefrom the amino acid sequence desired for expression in plants andproducing that nucleotide sequence through any number of means availablein the art. The preferred means uses phosphoramidite chemistries toconstruct short oligonucleotides that are each then linked together forform the full length sequence.

The invention also provides expression cassettes for use in plantscontaining sequences encoding all of, or an insecticidally activefragment of, or an amino acid sequence variant of, a Cry1Bb protein foruse in transforming plants to express said sequences. Nucleotidesequences comprising exemplary expression cassettes are referred toherein and as set forth in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, andSEQ ID NO:13. The subject invention also provides novel amino acidsequences comprising all or an insecticidally active fragment of aCry1Bb protein or equivalent as set forth in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14. Apolynucleotide sequence encoding an insecticidal fragment of a Cry1Bbcan be selected from the group of sequences consisting of from aboutnucleotide position 7 through about nucleotide position 1803 as setforth in SEQ ID NO:3, from about nucleotide position 2650 through aboutnucleotide position 4446 as set forth in SEQ ID NO:5, from aboutnucleotide position 3047 through about nucleotide position 4844 as setforth in SEQ ID NO:8, from about nucleotide position 1247 through aboutnucleotide position 3043 as set forth in SEQ ID NO:11, and from aboutnucleotide position 1658 through about nucleotide position 3454 as setforth in SEQ ID NO:13. Additionally, sequences encoding the amino acidsequences set forth in SEQ ID NO:2 from about amino acid position 2through about amino acid position 600, SEQ ID NO:4 from about amino acidposition 3 through about amino acid position 601, SEQ ID NO:7 from aboutamino acid position 3 through about amino acid position 601, SEQ IDNO:10 from about amino acid position 3 through about amino acid position601, SEQ ID NO:12 from about amino acid position 3 through about aminoacid position 601, and SEQ ID NO:14 from about amino acid position 3through about amino acid position 601 and that hybridize to the range ofnucleotide sequences as set forth above under stringent hybridizationconditions are within the scope of the present invention and compriseinsecticidally active fragments. Indeed, any peptide, for examplecomprising from about amino acid position 2 through about from as littleas amino acid position 600 up through amino acid position 1229 to 1230as set forth in SEQ ID NO:4 is considered to be within the definition ofan insecticidally active fragment. These proteins that are at least fromabout 598 to about 600 amino acids are sequences that are representativeof insecticidal fragments of the full length Cry1Bb insecticidal proteinexemplified from about amino acid position 2 through about amino acidposition 1228 or 1229 and are considered herein to be within the scopeof the present invention.

An additional embodiment consists of breeding together a firsttransgenic plant transformed to contain a first nucleotide sequenceencoding a first Bt insecticidal protein and a first herbicide tolerancemarker with a second transgenic plant transformed to contain a secondnucleotide sequence different from the first, encoding a second Btinsecticidal protein different from the first, and a second herbicidetolerance marker different from the first, to produce a third transgenicplant comprising a hybrid plant comprising both the first and the secondinsecticidal proteins and the first and second herbicide tolerancemarkers. The herbicide tolerance markers are selected from but notlimited to the group consisting of a gox enzyme, an antibioticresistance marker such as nptII, a glyphosate insensitive EPSPS enzyme,a basta resistance marker, and any other herbicide tolerance markerknown in the art, for example. The Bt insecticidal proteins can beselected from any of the known Cry1, Cry1, Cry3, Cry4, Cry5, Cry6, Cry9,Cry22, Cry33/34 binary toxins, as well as any other Bt insecticidalproteins known in the art such as VIP proteins and the like. Asexemplified herein, the first insecticidal protein may be a Cry1Bbprotein toxic to lepidopteran species, and the second insecticidalprotein need not be within the class of insecticidal proteins thatcontrols lepidopteran species, but instead can be within the class ofproteins known to be toxic to certain coleopteran insect species such asCry3 proteins, Cry5 proteins, various binary toxins known in the art,VIP proteins, and the like.

In fact, a first insecticidal resistance gene can be transformed into afirst plant along with a first selectable marker, such as a herbicidetolerance gene, to produce a first transgenic plant. A secondinsecticidal resistance gene different from the first can be transformedinto a second plant along with a second selectable marker, such as asecond herbicide tolerance gene, to produce a second transgenic plant.The first and the second transgenic plants can then be mated, assumingthe first and second plants are sufficiently related and capable ofbeing bred together, to produce a hybrid transgenic plant containingboth of the transgene alleles of the first transgenic plant and both ofthe transgene alleles of the second transgenic plant.

Other embodiments of the invention as set forth herein consist of plantscomprising the nucleotide sequences as set forth herein, plantscomprising nucleotide sequences which are substantially identical to thenucleotide sequences as set forth herein in which the sequence presentin plants comprises all or a part of the coding sequence for expressionof a Cry1Bb or amino acid sequence variant thereof in plants, said allor part of the coding sequence encoding a Cry1Bb or amino acid sequencevariant thereof sufficient to exhibit insecticidal activity to one ormore target insect plant pests of corn, cotton or soy and the like andwhich is no less toxic than the native full length Cry1Bb insecticidaltoxin. Plants, plant parts, progeny, and progeny or hybrid plantsderived from breeding with the recombinant plants of the presentinvention are encompassed as well, in particular those plants whichcontain one or more of the nucleotide sequences of the present inventionwhich encode a Cry1Bb protein or insecticidal portion of said protein.The sequences of the present invention are also intended to includenucleotide sequences exhibiting at least from about 75% to about 99% orgreater sequence identity with the sequences of the present invention.In addition, the sequences of the present invention are intended toinclude sequences that hybridize under stringent conditions to thesequences as set forth in the sequence listing herein.

A plant cell comprising a nucleotide sequence that functions forimproved expression in plants compared to a native Bt sequence encodinga Cry1Bb protein or insecticidal fragment thereof is contemplatedherein. Such plant cells are transformed with a nucleotide sequence thatcomprises a sequence selected from but not limited to the groupconsisting of from about nucleotide position 7 through about nucleotideposition 1803 as set forth in SEQ ID NO:3, from about nucleotideposition 2650 through about nucleotide position 4446 as set forth in SEQID NO:5, from about nucleotide position 3047 through about nucleotideposition 4844 as set forth in SEQ ID NO:8, from about nucleotideposition 1247 through about nucleotide position 3043 as set forth in SEQID NO:11, and from about nucleotide position 1658 through aboutnucleotide position 3454 as set forth in SEQ ID NO:13. Alternatively, acomplete Cry1Bb protein sequence can be expressed resulting in a proteinexhibiting an amino acid sequence substantially that as set forth in SEQID NO:4 from about amino acid position three through about amino acidposition 1229 or 1230. A method for preparing a transgenic plant cell asdescribed herein containing a nucleotide sequence encoding a full lengthCry1Bb or an insecticidally active fragment thereof is contemplated.Transgenic plants produced from the transformed cells are also withinthe scope of the present invention. In particular but not intending tobe limited by such disclosure, the plants including but not limited tomaize, wheat, sorghum, oat, barley, cotton, potato, tomato, soybean,canola, and fruit trees are specifically included within the scope ofthe present invention. Plants transformed with other nucleotidesequences encoding yet insecticidal proteins other than the insecticidalprotein of the present invention (Cry1Bb) can be bred to plantstransformed to contain only the Cry1Bb coding sequence, resulting in athird plant that is also a recombinant plant by virtue of it's heritage,and that exhibits improved insect resistance and tolerance to insectinfestation as a result of the presence of the two differentinsecticidal proteins. Furthermore, such progeny of a breeding can beeasily and simply identified by ensuring that each parental plant has aselectable marker present for conveying a double selection pressure uponthe hybrid plant produced as a result of the breeding of the two or moreplants. The result of course is a hybrid recombinant plant tat exhibitsat least one type of insect resistance (for example, a first insectresistance conveyed by the Cry1Bb gene, resistance to lepidopteranpests) but which may also exhibit a different insect resistance to thesame insect pests controlled by the Cry1Bb (which may be one or more ofan insecticidal protein including but not limited to a Cry1, a Cry2, aCry4, a Cry5, a Cry6, a Cry9, and a VIP1, VIP2, or a VIP3) or which mayexhibit a resistance to an entirely different class of plant insect pestspecies such as to Coleopteran species (which may require the use of oneor more of a Cry3A, a Cry3B, a Cry3C, a Cry22, ET70, TIC851, a binary Btinsecticidal protein toxin such as ET 33/34, ET80/76, or a CryP149B1).

Stringent conditions as defined herein include moderate to highstringency conditions which achieve the same, or about the same, degreeof specificity of hybridization as the conditions employed by theapplicants as exemplified herein. Examples of moderate and highstringency conditions are provided herein. Specifically, hybridizationof immobilized nucleotide sequences on means used for Southern blottingor on hybridization chips such as are well known in the art, forexample, with ³²P-labeled gene-specific probes or primers can beperformed by standard methods (Sambrook, Fritsch, & Maniatis; MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, NY 1989). In general, hybridization and subsequent washes can becarried out under moderate to high stringency conditions that allow fordetection of target sequences with homology to the exemplified toxingenes. For double-stranded nucleotide probes, hybridization can becarried out D overnight at 20-25 C below the melting temperature (Tm) ofthe DNA hybrid in 6×SSPE, 5× denhardts solution, 0.1% SDS, 0.1 mg per mldenatured nucleotide probe. The melting temperature can be described bythe following formula as set forth in Beltz et al. (1983, Methods inEnzymology, 100:266-285, Wu, Grossman, and Moldave Eds., Academic Press,NY)Tm=81.5 C+16.6 Log[Na^(+])+0.41 (% G+C)−0.61 (% formamide)−600/length ofduplex in base pairs.Washes are typically carried out as follows:(1) two washes at room temperature for about fifteen (15) minutes in1×SSPE, 0.1% SDS (low stringency wash), followed by(2) one wash at Tm −20 C for about fifteen (15) minutes in 0.2×SSPE,0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20 C below the melting temperature (Tm) of the hybrid in 6×SSPE,5× Denhardts solution, 0.1% SDS, 0.1 μg per ml denatured probe. The Tmfor oligonucleotide probes can be described by the following formula asset forth in Suggs et al. (1981, ICN-UCLA Symp. Dev. Biol. UsingPurified Genes, 23:683-693, D. D. Brown Ed., Academic Press, NY):Tm(C)=2(No. T&A base pairs)+4(No. G&C base pairs).Washes using oligonucleotide probes can be carried out as describedabove. For probe sequences of greater than about seventy (70)nucleotides in length, a low stringency condition for hybridizationwould be equivalent to suspension in either 1× or 2×SSPE at atemperature from about room temperature to about 42 C. A moderatestringency condition for hybridization would be equivalent to suspensionin from about 0.2× to about 1×SSPE at a temperature of about 65 C. Ahigh stringency hybridization condition would be equivalent tosuspension in from about 0.01× or less to about 0.1×SSPE at atemperature of about 65 C.

The amino acid sequences of the present invention are intended toinclude analogs or homologs or other related amino acid sequences whichare sufficient to exhibit insecticidal bioactivity at least equivalentto that exhibited by the native Cry1Bb full length protein, including atleast amino acid sequences which are from about 95% identical to about99% identical or greater in amino acid sequence to the sequenceexhibited by the amino acid sequence as set forth in SEQ ID NO:2 or SEQID NO:4.

Another embodiment of the present invention provides a method fortransforming a plant to express a Cry1Bb protein or amino acid sequencevariant or insecticidally active fragment thereof.

Still another embodiment provides methods for detecting the presence ofa sequence disclosed herein in the present invention in a plant, plantcell, or biological sample. The detection of a nucleotide sequenceexpressing Cry1Bb protein in a plant would be diagnostic for a plantcontaining said nucleotide sequence within its nuclear or plastidgenome. Furthermore, antibodies which specifically bind to a Cry1Bbprotein are set forth in the examples. Such antibodies are exemplary foruse in detecting the presence of a plant expressing all or a part of aCry1Bb protein, and for detecting a plant comprising a nucleotidesequence that encodes a Cry1Bb protein. The detection of Cry1Bb proteinusing immunological methods would be diagnostic for a plant comprisingany of the nucleotide sequences set forth herein which express a Cry1Bbprotein or equivalent.

A biological sample consisting primarily of a plant containing one ormore of the nucleotide sequences of the present invention is believed tobe within the scope of the present invention. A biological samplederived from a plant, a plant tissue, or a plant seed, wherein thesample contains a nucleotide sequence that is or is complementary to asequence selected from but not limited to a group of sequencesconsisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, andSEQ ID NO:13, in which the sequence is detectable in the sample using anucleic acid amplification or nucleic acid hybridization method, iscontemplated specifically herein to be within the scope of the presentinvention. A biological sample is intended to include a plant, planttissue, or plant seed that contains one or more of the nucleotidesequences exemplified herein, as well as products produced from suchplant, plant parts, or plant seeds including but not limited to flourderived from soy or wheat or barley or oat or potato or corn, soy orcorn meal, corn syrup, corn or soy or canola oils, corn starch, andcereals manufactured in whole or in part to contain corn, soy, wheat,barley, oat, flax or other cereal plant by-products that contains adetectable amount of one or more of the nucleotide sequences of thepresent invention, wherein the nucleotide sequences are detectable insaid biological sample or extract using any nucleic acid amplificationor nucleic acid hybridization method.

Similarly, a kit for detecting the presence of Cry1Bb protein in asample is contemplated by the instant invention. The kit would provide atest reagent containing a Cry1Bb positive control sample along with anegative control, antibodies which bind specifically to a Cry1Bbprotein, and the reagents necessary to carry out a determinativereaction with the control samples as well as an unknown sample suspectedof containing an immunologically detectable amount of a Cry1Bb protein,packaged together in said kit with instructions for use. Antibodies thatbind specifically to Cry1Bb and not to other Bt insecticidal proteinsare particularly suited for use in kits based on immunological methodsand are believed to be within the scope of this invention. A similar kitfor detecting the presence of a nucleotide sequence as set forth herein,encoding at least an insecticidally active Cry1Bb protein or fragmentthereof, is specifically contemplated herein. Exemplary are nucleotidesequences which could be used as probes for detecting a sufficientamount of a nucleotide sequence derived from a polynucleotide sequenceencoding a Cry1Bb protein, or nucleotide sequences in the form of primerpairs which could be used as amplification primers for producing all ora part of the Cry1Bb encoding nucleotide sequences encompassed by thisdisclosure, for example by using thermal amplification methods wellknown in the art. Such primers or probes along with positive andnegative control samples packaged together in a kit, or packagedseparately, and distributed with the necessary reagents for completing ahybridization or amplification reaction to detect all or a part of theCry1Bb encoding nucleotide sequences encompassed by the instantinvention, along with instructions for use are specifically contemplatedherein.

The regulation of expression of the sequences of the present inventioncan be accomplished in a number of different ways. One means would be torely on the particular operably linked promoter sequence which drivesexpression of the transgene to effectively regulate the expression ofthe Cry1Bb protein. Generally, this means results in the expressionbeing determined by the type of linked promoter, i.e., a promoter thatis temporally or spatially regulated within the cell or tissue typewithin the plant by factors that are beyond the control of the skilledartisan. Promoters such as these are generally either “on” at all timesthroughout the growth and development of the plant. Other promoters maybe “enhanced” in that they are on at characteristically prominent times,for example, only when the plant is flowering, or only when the plant isdeveloping from an embryo within the germinating seed into a shoot or ahairy root, or only substantially within the root, etc. The range ofpromoters available for such temporal and spatial expression within aplant, and more particularly, within a plant type, is too numerous todiscuss here. However, using antisense technologies, the transcribedmessenger RNA can be regulated in such a way as to elevate the level ofprotein produced within a plant or to decrease the level of proteinproduced in a plant. One particularly useful means for regulating thelevel of messenger RNA in a cell is RNAi technology exemplified in WO01/75164 (Tuschl et al.), WO 99/61631 (Heifetz et al.), WO 99/53050(Waterhouse et al.), WO 99/49029 (Graham et al.), WO 99/32619 (Fire etal.), WO 98/05770 (Werner et al.). A summary of the known RNAitechnology can be found at Lau et al. Scientific American August 2003pp. 3441). The expression of the constructs exemplified herein in plantscan be subjected to these means for regulating and modulating theexpression of the proteins expressed therefrom.

3.0 DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 represents a native Bacillus thuringiensis nucleotidesequence encoding a native Cry1Bb protein as set forth in Donovan etal., U.S. Pat. No. 5,679,343, and described therein as cryET5 encodingCryET5.

SEQ ID NO: represents the deduced full length amino acid sequencetranslation of a native Cry1Bb protein from the open reading frameidentified as being present in the nucleotide sequence of SEQ ID NO:1.

SEQ ID NO:3 represents a non-naturally occurring or synthetic nucleotidesequence exhibiting, when compared to the native coding sequence,improved in planta levels of expression of a Cry1Bb variant protein, andwhich encodes an amino acid sequence variant of a Cry1Bb protein. SEQ IDNO:4 represents the deduced amino acid sequence translation of thenucleotide sequence as set forth in SEQ ID NO:3 encoding a Cry1Bb aminoacid sequence variant.

SEQ ID NO:5 represents a non-naturally occurring nucleotide sequencecomprising an expression cassette comprising the operably linkedelements P-FMV: L-Os.βtub : I-Os.PAL: cry1Bb1 variant: T-Os.Ldh(corresponding to a figwort mosaic virus promoter, a rice pal geneintron, a synthetic nucleotide sequence encoding a Cry1Bb variantprotein, and a rice lactate dehydrogenase termination andpolyadenylation sequence) present as set forth in both pMON33731 andpMON33733, exhibiting improved in planta levels of expression of aCry1Bb variant protein.

SEQ ID NO:6 represents the amino acid sequence translation of anucleotide sequence as set forth in SEQ ID NO:5 from about nucleotideposition 526 through about nucleotide position 1317 encoding an NptIIprotein used primarily in the applications as set forth herein as aselectable marker for identifying plant cells and plants transformed bya vector or sequence containing the nptII gene linked to some other geneof interest.

SEQ ID NO:7 represents the amino acid sequence translation of anucleotide sequence as set forth in SEQ ID NO:5 from about nucleotideposition 2644 through about nucleotide position 6333 encoding a Cry1Bbamino acid sequence variant.

SEQ ID NO:8 represents a non-naturally occurring or synthetic nucleotidesequence comprising an expression cassette comprising the operablylinked elements P-FMV: L-Os.βtub : I-Os.PAL : TP-Zm.rbcs: cry1Bb1variant: T-Os.Ldh (corresponding to the following operably linkedgenetic elements: a figwort mosaic virus promoter, a rice pal geneintron sequence, a sequence encoding a corn or maize ribulosebis-phosphate carboxylase synthase small subunit chloroplast targetingpeptide (rbcs) interrupted by a small intron native to the cornsequence, a coding sequence encoding a Cry1Bb amino acid sequencevariant, and a rice lactate dehydrogenase transcription termination andpolyadenylation sequence) present in both pMON33732, pMON33734,pMON33750, and pMON40213 (except that a sequence encoding a glyphosatetolerant CP4 EPSPS is present in place of the NptII coding sequence inpMON33750 and pMON40213) that exhibits enhanced in planta expression ofthe plastid targeted Cry1Bb amino acid sequence variant.

SEQ ID NO:9 represents the amino acid sequence translation of anucleotide sequence as set forth in SEQ ID NO:8 from about nucleotideposition 526 to about nucleotide position 1317 encoding an NptII proteinused primarily in the applications as set forth herein as a selectablemarker for identifying plant cells and plants transformed by a vector orsequence containing the nptII gene linked to some other gene ofinterest.

SEQ ID NO:10 represents the amino acid sequence translation of thenucleotide sequence as set forth in SEQ ID NO:8 from about nucleotideposition 3041 through about 6730 encoding a plastid targeted Cry1Bbamino acid sequence variant.

SEQ ID NO:11 represents a non-naturally occurring or syntheticnucleotide sequence comprising an expression cassette comprising theoperably linked elements P-e35S : L-TaCab : 1-Os.Act1 : cry1Bb1 variant: T-Ta.Hsp17 (corresponding to the following operably linked elements:enhanced cauliflower mosaic virus ³⁵S promoter, a 5′ untranslated wheatchlorophyll a/b binding protein gene leader sequence, a rice actinintron sequence, a Cry1Bb amino acid sequence variant coding sequence,and a wheat hsp17 heat shock gene transcription termination andpolyadenylation sequence) present in pMON40227 exhibiting enhanced inplanta expression of a Cry1Bb amino acid sequence variant.

SEQ ID NO:12 represents the amino acid sequence translation of thenucleotide sequence as set forth in SEQ ID NO:11 from about nucleotideposition 1241 through about nucleotide position 4930 encoding a Cry1Bbamino acid sequence variant.

SEQ ID NO:13 represents a non-naturally occurring nucleotide sequencecomprising an expression cassette comprising the operably linkedelements P-e35S : L-Ta.Cab : I-Os.Act1: TP-Zm.rbcs: cry1Bb1 variant :T-Ta.Hsp17 (corresponding to the following operably linked elements:enhanced cauliflower mosaic virus promoter, a 5′ untranslated wheatchlorophyll a/b binding protein gene leader sequence, a rice actinintron sequence, a sequence encoding a corn or maize ribulosebis-phosphate carboxylase synthase small subunit chloroplast targetingpeptide (rbcs) interrupted by a small intron native to the cornsequence, a synthetic sequence encoding a Cry1Bb1 amino acid sequencevariant, and a wheat heat shock Hsp17 protein transcription terminationand polyadenylation sequence) present in pMON40228 exhibiting improvedin planta expression of a plastid targeted Cry1Bb amino acid sequencevariant.

SEQ ID NO:14 represents the amino acid sequence translation of thenucleotide sequence as set forth in SEQ ID NO:13 from about nucleotideposition 1652 through about nucleotide position 5341 encoding a plastidtargeted Cry1Bb amino acid sequence variant.

4.0 DETAILED DESCRIPTION OF THE INVENTION

The subject matter encompassed by the instant invention includescompositions and methods for use in the control of plant infestation byinsect pest species, and in particular, control of infestation by larvaeof various lepidopteran insect pest species susceptible to or controlledby ingestion of insecticidally effective amounts of a Bacillusthuringiensis Cry1Bb protein. More specifically, nucleotide sequenceswhich have been designed for enhanced and/or improved expression ofCry1Bb pesticidal toxin in plant cells and in plant tissue areencompassed by the instant invention, including full length Cry1Bb, coretoxin or tryptic fragments of Cry1Bb, less than full length Cry1Bbtoxin, and fragments which are smaller in mass than the core or trypticfragment but which retain insecticidal bioactivity to one or more insectspecies which are normally inhibited or killed by ingestion of fulllength Cry1Bb toxin.

Reference to “full length” is intended to include but is not intended tobe limited to a nucleotide sequence which encodes all of the nativeCry1Bb toxin or an amino acid sequence variant of the Cry1Bb toxin whichretains bioactivity no less than that observed for controlling at leastone insect pest species normally controlled by the native Cry1Bb toxin.The term “full length” is also intended to refer to the form of theCry1Bb toxin produced or expressed from a nucleotide coding sequence ofthe instant invention. A full length Cry1Bb toxin protein will berecognized by one skilled in the art to be a protein substantiallyidentical in length of amino acid sequence to the native Cry1Bb proteinexpressed from the native gene in Bacillus thuringiensis. A typical Cry1protein is comprised of a toxin domain positioned at the amino terminalend of the Cry1 protein sequence and a protoxin domain linked to andpositioned at the carboxy-terminal end of the toxin domain. The toxindomain is typically further comprised of three sub-domains described inthe literature as domain I, domain II, and domain III, the preciselocation of the region defining either end of each of these sub-domainsbeing somewhat arbitrary but generally based on degrees of homology,identity, or similarity between amino acid sequences of other Cry1proteins within a particular class of Cry1's. Generally, domain I ispositioned at the amino terminal end of the toxin domain and is linkedat its carboxy terminal end to the amino terminal end of domain II,which is in turn linked at its carboxy terminal end to the aminoterminal end of domain III. Sub-domains of the toxin domain have alsobeen identified in the art by reference to amino acid sequence positionalong the length of a given Cry1 protein. Interestingly, Cry2 and Cry3toxin proteins exhibit this structural similarity, although the degreeof identity between sub-domains when comparing Cry1's to either Cry2 orCry3 proteins is more divergent. An insecticidal fragment of any of theproteins of the present invention will be recognized by those of skillin the art as any amino acid sequence which is greater than about 95%identical at the amino acid sequence level to the Cry1Bb proteins of thepresent invention and which retain insecticidal bioactivity no less thanthat of the full length Cry1Bb1 (CryET5) native protein. Preferredinsecticidal fragments of the present invention include from about aminoacid sequence position one through about amino acid position 600, orthrough about amino acid position 643, or of the sequences as set forthin either SEQ ID NO:2 or SEQ ID NO:4, or amino acid sequences which aresubstantially the same as those sequences or within a range of about 95%sequence identity at the amino acid sequence level to the amino acidsequence of the first 643 or so amino terminal amino acids.

A number of insecticidally useful chimeric proteins have been disclosedwhich are comprised of combinations of sub-domains from differentBacillus thuringiensis insecticidal crystal protein toxins. For example,Fischhoff et al. described a chimeric toxin formed from linking domainsI and II of a first Cry protein, Cry1Ab, to domain III of a second Cryprotein, Cry1Ac, which exhibited insecticidal bioactivity at least asgreat as the insecticidal bioactivity of either of the parent toxins(U.S. Pat. Nos. 5,500,365, 5,880,275). Perlak et al. also described agene identical to that of Fischhoff et al. (BioTechnol. 1990,8:939-943). Bosch et al. also disclosed chimeric toxins comprising avariety of formulations consisting of domains I and II of a first Cryprotein linked to domain III of a Cry protein different from the first,and noted that it was unpredictable to determine which, if any, wouldfunction in providing insecticidal activity at least as great as that ofthe parent toxins (WO95/06730). Malvar et al. have also disclosedchimeric amino acid sequences formed from the operable linkage, fromamino to carboxy terminal ends, of domain I of a first Cry protein withdomain II and domain III of a second Cry protein which is different fromthe first Cry protein; and domain I and domain II of a first Cry proteinwith domain III of a second Cry protein which is different from thefirst (U.S. Pat. Nos. 6,017,534, 6,110,464, 6,221,649, and 6,242,241).It is likely that other such chimeric toxins could also be constructed,but it would not be known which if any of the chimeric toxins wouldexhibit insecticidal activity, and whether any insecticidal activitywould be an improvement over any of the native toxins from which thesub-domains were selected for incorporation into the chimera.

The nucleotide sequences of the present invention exhibit individualnucleotides and sequences of nucleotides that are different incomposition relative to the corresponding coding sequences containedwithin the native Bacillus thuringiensis sequence encoding Cry1Bb. Suchdifferences include reductions in the overall adenosine and thymidinecomposition of the nucleotide sequence compared to the native Btsequence; a modified preference for various codons which, in Bacillusthuringiensis, would otherwise be preferred for use, in particular withreference to the third base position for each codon such that for aminoacids for which there are at least two or more codons, a preference foruse of those codons which do not have an A or a T in the third baseposition; and an overall guanosine and cytosine composition from about50% to about 60% or more; and an overall reduction in the appearance ofputative polyadenylation sequences as set forth in Fischhoff et al.(U.S. Pat. No. 5,500,365). Such nucleotide sequences of the presentinvention which encode all or an insecticidally active fragment of aCry1Bb protein exhibit an improved level of expression in plantscompared to the native Cry1Bb protein sequence obtained from Bacillusthuringiensis, particularly when operably linked at least to a plantfunctional promoter and a plant functional transcription termination andpolyadenylation sequence, or when operably linked to a promoterfunctional in a plant chloroplast and targeted for expression within theplant chloroplast. The sequences of the present invention are thereforeparticularly well-suited for optimized expression in plants, and can beused by those skilled in the art to transform plant cells, regeneraterecombinant plants from the transformed plant cells, and to obtaincommercially useful plants which express insecticidally effectiveamounts of all or an insecticidally active fragment of a Cry1Bb proteinfor inhibiting insect infestation of the plant. The words “plantfunctional”, with reference to nucleotide sequences, are intended toindicate that the particular sequence referred to, such as a promoter,an intron, an untranslated leader, a transcription initiation sequence,a coding sequence, and/or a transcription termination andpolyadenylation sequence operates in a plant with the molecular andcellular machinery involved in transcription and translation and posttranslation in a way which is intended to bring about the production ofan amino acid sequence encoded by the coding sequences to which theplant functional sequences are linked.

In one embodiment, the invention provides nucleotide sequences forexpression in plants that encode a Cry1Bb toxin or an insecticidallyactive fragment of a Cry1Bb toxin that is active against lepidopteraninsects. These nucleotide sequences include genes designed forexpression in plants, and these genes can be selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, andSEQ ID NO:13.

In another embodiment, the invention also provides nucleotide sequencesfor expression in plants that encode a Cry1Bb protein or fragmentthereof toxic to lepidopteran insect pests that typically infestcommercial crops. Such protein sequences include SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID. NO:14. Peststypically infesting commercial crops are described herein, but includeat least armyworms, rootworms, boll worms, loopers, earworms, bud worms,and stem borers.

The subject invention provides nucleotide sequences encoding aninsecticidally active fragment of a Cry1Bb protein linked to a protoxindomain of a Cry1 toxin other than a Cry1Bb toxin. Conversely, thepresent invention also provides a novel nucleotide sequence encoding aCry1Bb protoxin domain which can be used for constructing a nucleotidesequence encoding a full length Cry1 related toxin in which the toxindomain is other than a Cry1Bb toxin domain. Additionally, the presentinvention provides nucleotide sequences encoding amino acid sequencescorresponding to sub-domains of a Cry1Bb toxin fragment, and moreparticularly corresponding to domain I, domain II, and domain III of theCry1Bb toxin fragment, which can be used to construct novel toxinscomprising all or any part of each of these sub-domains of the Cry1Bbtoxin domain amino acid sequences.

In another embodiment, the present invention provides nucleotidesequences that express a Cry1Bb toxin that is less than full lengthcompared to the full length Cry1Bb toxin produced by Bacillusthuringiensis. Such nucleotide sequences encoding a less than fulllength Cry1Bb amino acid sequence typically do not contain all or aportion of the protoxin fragment of the full-length native Cry1Bbprotein. Nucleotide sequences encoding a less than full-length Cry1Bbamino acid sequence could be used for the production of nucleotidesequences which encode a fusion or chimeric protein toxin.

One example of a nucleotide sequence which has been designed forenhanced and/or improved expression of Cry1Bb pesticidal toxin in plantcells and in plant tissue is SEQ ID NO:3 which substantially encodes anative Cry1Bb amino acid sequence. The difference between the amino acidsequence encoded by SEQ ID NO:3 and the native Cry1Bb sequence residesin the amino terminus of the peptide sequence. The native codingsequence (SEQ ID NO:1) initiates with the codon “ttg”, which upontranslation of the corresponding position in the mRNA corresponding tothe transcription product produced from the cry1Bb gene in Bacillusthuringiensis results in the incorporation of a leucine amino acidresidue at the first amino acid sequence position in the native Cry1Bbprotein (SEQ ID NO:2 herein, and referenced in Donovan et al., U.S. Pat.No. 5,679,343). The second and third amino acid residues comprising thenative Cry1Bb sequence are threonine and serine respectively. While theplant functional coding sequences of the present invention encode anamino acid sequence identical to the composition of the native Cry1Bbamino acid sequence corresponding to the amino acid sequence of thenative Cry1Bb from position two (threonine) through at least theinsecticidal core sequence of the toxin, the first two codons in thesynthetic gene (at least with reference to SEQ ID NO:3 and its aminoacid sequence translation at SEQ ID NO:4) encode for the incorporationof the amino acid residues methionine and alanine respectively at aminoacid sequence positions one and two in the Cry1Bb proteins encoded bythe nucleotide sequences intended for use in plants.

An insecticidal toxin protein expressed from the nucleotide sequences ofthe present invention comprises at least a core toxin fragmentcomprising and corresponding to approximately the first six-hundred andforty-three (643) amino acids of the native Cry1Bb protein as set forthin SEQ ID NO:2, or corresponding to approximately the first six-hundredforty four (644) amino acids of the Cry1Bb protein encoded by thesynthetic nucleotide sequences of the present invention, as exemplifiedby the sequence as set forth in SEQ ID NO:4. However, a toxin proteinproduced from the nucleotide sequences of the present invention, whichis substantially identical in amino acid sequence to a native Cry1Bbcore toxin fragment, and which retains insecticidal activity to one ormore lepidopteran pests previously demonstrated to be susceptible to atleast the core toxin fragment, although consisting of an amino acidsequence slightly shorter than or slightly longer than the native coretoxin but retaining no less insecticidal bioactivity than the nativecore toxin fragment, is also considered to be within the scope of theinvention. SEQ ID NO:3, for example, comprises a synthetic nucleotidesequence which encodes an amino acid sequence variant of a Cry1Bbprotein which retains lepidopteran insecticidal bioactivity equivalentto or greater than the bioactivity of the native Cry1Bb protein. SEQ IDNO:3 also encodes a core toxin fragment comprising from about amino acidposition 1 through about amino acid position 644 as set forth in SEQ IDNO:4, corresponding substantially to a Cry1Bb core insecticidal crystalprotein fragment, which retains bioactivity equivalent to or greaterthan that of the native Cry1Bb protein as set forth in SEQ ID NO:2. Itis shown herein that a Cry1Bb fragment as set forth in SEQ ID NO:4 whichcorresponds to an amino acid sequence of from about 1 through aboutamino acid position 640 is sufficient to provide bioactivity equivalentto or greater than that of a native Cry1Bb protein. This wouldcorrespond to a native core toxin fragment of about the firstsix-hundred thirty nine (639) amino acids as set forth in SEQ ID NO:2.This would correspond to a native core toxin fragment of about the firstsix-hundred and thirty nine (639) amino acids as set forth in SEQ IDNO:2.

The overall amino acid sequence alignment of the native Cry1Bb to otherknown native Cry1 proteins provides insight into the relevantbreakpoints between the sub-domains within the toxin fragment, and therelative breakpoint between the toxin domain and the protoxin domain ofthe native Cry1Bb full length protein. The native Cry1Bb amino acidsequence is comprised of (a) domain I from about amino acid one (1)through about amino acid two-hundred eighty-eight (288) as set forth inSEQ ID NO:2, corresponding to nucleotide position from about one (1)through about nucleotide position eight-hundred sixty-four (864) as setforth in SEQ ID NO:1; (b) domain II from about amino acid two-hundredeighty-nine (298) through about amino acid four-hundred ninety-six (496)as set forth in SEQ ID NO:2, corresponding to nucleotide position fromabout eight-hundred sixty-five (865) through about nucleotide positionfourteen-hundred eighty-eight (1488) as set forth in SEQ ID NO:1; (c)domain III from about amino acid four-hundred ninety-seven (497) throughabout amino acid six-hundred forty-three (643) as set forth in SEQ IDNO:2, corresponding to nucleotide position from about fourteen-hundredeighty-nine (1489) through about nucleotide position nineteen-hundredtwenty-nine (1929) as set forth in SEQ ID NO:1; and (d) the protoxindomain from about amino acid six-hundred forty-four (644) through aboutamino acid twelve-hundred twenty-nine (1229) as set forth in SEQ IDNO:2, corresponding to nucleotide position from about nineteen-hundredthirty (1930) through about nucleotide position thirty-six-hundredeighty-seven (3687) as set forth in SEQ ID NO:1.

The overall sequence of the amino acid variant Cry1Bb protein sequencesdisclosed herein resembles the native amino acid sequence, however thepositions of the breakpoints for the sub-domains and the protoxin totoxin domain junction is shifted up one additional numerical valuerelative to the modification of the initiation sequences utilized forexpression in planta, for example, as set forth in SEQ ID NO:4. Thesynthetic coding sequence is comprised of codons at nucleotide positionsone through six (1-6) encoding an amino terminal MET-ALA di-peptiderepresenting the first two amino acids in the amino acid sequence as setforth in SEQ ID NO:4, for example, engineered into the Cry1Bb sequenceencoded by the synthetic sequences of the present invention. These twoamino acid residues replace or are substituted for the native aminoterminal LEU residue, therefore adding an additional amino acid residueat the amino terminus of the encoded Cry1Bb variant, resulting in theup-shift in position of the amino acid residues corresponding to theapproximate breakpoints between the sub-domains I, II and III, and thetoxin to protoxin domains.

Nucleotide sequences of the present invention which encode only an aminoacid sequence corresponding to a Cry1Bb core toxin fragment are expectedto be efficiently expressed in planta, however in some plants the coretoxin fragment produced from expression from a nucleotide sequence whichis less than full length when compared to the native Cry1Bb codingsequence may result in plants which exhibit physiologicalcharacteristics which are undesireable. In that event, it is likely thatthe construction of a nucleotide sequence encoding a Cry1 protoxindomain operatively linked to the coding sequence of the Cry1Bb coretoxin fragment would stabilize the expression of the Cry1Bb protein.Therefore, fusion peptides of a Cry1Bb core toxin fragment to a protoxindomain of any other Cry1 toxin is contemplated as a specific embodimentof the invention. It is apparent that there can be some overlap betweenthe nucleotide sequences encoding a Cry1Bb protein that is less thanfull length and the nucleotide sequences encoding the protoxin portionsof Cry1 proteins.

The nucleotide sequences of the present invention, with reference to thesequence encoding the Cry1Bb or amino acid sequence variants of Cry1Bbare comprised of from about 50% to about 65% GC content, or from about55 to about 64% GC content, or from about 60 to about 64% GC content, orabout 64% GC content. One skilled in the art will recognize that thisrange of GC % is highly variable due to the redundancy of the geneticcode, and so the GC % of a nucleotide sequence encoding a full lengthCry1Bb or an insecticidal Cry1Bb amino acid sequence variant orinsecticidal fragment thereof would range from about 46% or 48% GC onthe low end up to about 60% or 65% GC or more depending upon the natureof the host cell in which expression is desired. This range is achievedwithout sacrificing substantially improved levels of expression inplanta. The nucleotide sequences of the present invention correspond tosequences prepared by observing the amino acid sequence of the Cry1Bbnative amino acid sequence and deducing the amino acid sequence intendedfor expression in planta. Substantially, the sequences of the presentinvention were prepared according to the methods as set forth in Brownet al. (U.S. Pat. No. 5,689,052) except that the starting material wasnot the native Cry1Bb coding sequence but was the native Cry1Bb aminoacid sequence, and no partial sequences were prepared, but instead anentirely new nucleotide sequence was prepared using computer algorithms.The computer generated sequence was provided to a nucleotide synthesisservice provider that completely synthesized the new sequence encodingthe Cry1Bb amino acid sequence variants, confirmed the new sequence bysequencing the synthetic coding sequence in both directions, andprovided the newly synthesized sequence in a cassette in a plasmid, thecassette flanked on either end by restriction endonuclease recognitionsites engineered into the terminal ends of the synthetic sequence forthe purpose of convenience in further manipulations designed for addingplant functional promoter sequences, plant functional intronicsequences, untranslated plant functional leader sequences, and plantfunctional 3′ transcription termination and polyadenylation sequences.

The DNA constructs of the present invention comprise fully syntheticstructural coding sequences that enhance the performance of the sequencein plants. In a particular embodiment of the present invention, theenhancement method has been applied to design fully synthetic codingsequences encoding Cry1Bb variant insecticidal proteins. The structuralgenes of the present invention may optionally encode a fusion proteincomprising an amino-terminal plastid or chloroplast transit peptide or asecretory signal sequence.

It should be apparent to one skilled in the art that the nucleotidesequences of the present invention can be constructed through severalmeans. The nucleotide sequences of the present invention can bepartially or even entirely constructed using a gene sequence synthesizerusing, for example, phosphoramidite or related chemistries to linkindividual nucleotides into a polynucleotide sequence. Sequences whichrepresent partial sequences encoding parts or fragments of the Cry1Bb orvariant sequence can be inserted into the native sequence, or can beused as primers for linking the synthetic sequence to the nativesequence so long as there is sufficient overlap or complementaritybetween all or a part of the synthetic sequence. The exemplifiedsequences can also be obtained or constructed by modifying the nativegene encoding a Cry1Bb protein, for example, by point mutation orsequence replacement, and in particular using thermal amplification orother DNA synthesis and primer extension methodologies.

The nucleotide sequences of the present invention can also be used toform complete genes that encode proteins or peptides in a desired hostcell. For example, those of skill in the art will recognize that thenucleotide sequences of the present invention can be illustrated in thesequence listing without termination codons in frame with and at theterminus of the coding sequence for the Cry1Bb protein. Nucleotidesequences encoding the Cry1Bb protein or variants thereof can be placedunder the control of a promoter sequence for expression of the Cry1Bbprotein in any host cell of interest. Methods and examples of thesemodifications are readily identifiable in the art.

The nucleotide sequences of the present invention can exist in eithersingle or double stranded form. Double stranded forms are comprised ofone strand that is complementary to the other strand and vice versa. Thecoding strand is referred to in the art as the strand or sequencecontaining the series of codons or base triplets that can be read as anopen reading frame (ORF) to form a protein or peptide of interest.Expression of the protein necessarily involves transcription of thecomplementary or non-coding strand to produce a messenger RNA sequencewhich corresponds to the coding strand, which is used by the host cell'stranslational machinery as the template for the assembly of amino acidsinto a linear sequence corresponding to the sequence of the amino acidsequences of the present invention. Therefore, the subject inventionincludes the use of either the exemplified nucleotide sequences as setforth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ IDNO:13 and the corresponding complementary strands or sequencescomplementary to the exemplified nucleotide sequences. RNA moleculesthat are functionally equivalent to the exemplified nucleotide sequencesare included in the subject invention.

It is specifically intended that the present invention includesequivalent and variants of the nucleotide sequences and amino acidsequences of the present invention, including but not limited tomutants, fusions, chimeras, truncations, fragments, and smaller orshorter genes and amino acid sequences. In particular, it is importantto recognize that the intended sequences and variants thereof exhibitthe same or similar characteristics relating to expression of toxins inplants, as compared to those specifically disclosed herein. As usedherein, variants and equivalents includes reference to sequences whichhave nucleotide or amino acid substitutions, deletions whether internaland or terminal, additions, or insertions which do not materially affectthe expression of the subject gene or genes or expression cassettes, andthe resultant pesticidal activity in plants. Fragments that retainpesticidal activity are also included in this definition. Thus,nucleotide sequences that are smaller or shorter than those specificallyexemplified are included in the subject invention, so long as thenucleotide sequence encodes a toxin that exhibits insecticidalbioactivity.

Genes and expression cassettes can be modified, and variations of thesemodifications can be readily constructed, using methods well known inthe art. For example, methods for making individual nucleotide sequencechanges described in the art as point mutations are well known in theart. In addition, commercially available nucleases are available for usein constructing sequences that are redacted in sequence in comparison tothe nucleotide sequence that was used as the starting material. Suchenzymes can be used to systematically excise various lengths of sequencefrom one end or the other of a linear nucleotide sequence.

In addition, restriction endonucleases can be used to constructfragments of sequences that can be moved into other sequences forconstruction of chimeras, variants, and modified sequences of thepresent invention.

It is apparent that equivalent genes will encode amino acid sequencescorresponding to a Cry1Bb protein or variant thereof, and the proteinwill exhibit high amino acid sequence identity or homology with thenative Cry1Bb protein or insecticidal amino acid sequence deletions,truncations, or variants thereof. The amino acid sequence homology willbe the highest in the critical regions of the toxin that account forbiological activity or are involved in the determination forthree-dimensional configuration of the protein. For example, it is wellknown that the Cry1, Cry2, and Cry3 proteins fold into a threedimensional globular structure, and that each of the domains referred tohereinabove comprise each of the three globular domains which comprisethe overall globular structure of these proteins. Particular folds,turns, or beta-sheet configurations require specific compositions ofamino acid sequences to properly effectuate the overall intendedinsecticidal configuration and activity of the protein molecule.Incorporation of charged residues in regions in which there werepreviously no charged residues is likely to disrupt the configuration ofthe region, and likely therefore to disrupt the configuration of theoverall protein, resulting in a loss of activity and the like. It iswell known that each of the twenty naturally and most commonly occurringamino acids may be placed into various classes characterized asnon-polar, uncharged-polar, basic, and acidic. Conservativesubstitutions, i.e., replacement of an amino acid of one class by anamino acid of the same type or class, fall within the scope of thesubject invention so long as the substitution does not materially alterthe exhibited biological activity of the Cry1Bb protein. Suchconservative substitutions that are possible are well known in the artand can be readily identified using any biochemistry text book orequivalent resource. Nucleotide sequences encoding insecticidalfragments or even full length Cry1Bb proteins that hybridize to thenucleotide sequences as set forth herein under stringent conditions arebelieved to be within the scope of the present invention, in particularif the sequences are intended for use in expression of the Cry1Bbprotein in plants. In particular, sequences that are from about 75% toabout 80% identical in nucleotide sequence, or from about 80% to about90% identical in nucleotide sequence, or from about 90% to about 99%identical in nucleotide sequence to the sequences of the presentinvention encoding Cry1Bb as set forth in SEQ ID NO:3, SEQ ID NO:5, SEQID NO:8, SEQ ID NO:11, and SEQ ID NO:13 are believed to be within theembodiments of the present invention.

In some cases non-conservative substitutions can be made whichsurprisingly increase the insecticidal activity, and do not reduce thein planta expression of the nucleotide sequence encoding the modifiedamino acid sequence variant Cry1Bb protein.

As used herein, reference to the word isolated nucleotide sequences and,or purified insecticidal toxin refers to these molecules when they arenot associated with the other molecules with which they would be foundin naturally occurring biological systems. For example, an isolatedand/or purified nucleotide sequence encoding a Cry1Bb insecticidalprotein or insecticidal fragment thereof would include its use in plantsand in kits designed for use in detection of the molecules in biologicalsamples. Such biological samples would include whole plants and or plantcells transformed to express a Cry1Bb protein or immunologically relatedCry1Bb amino acid sequence variant, nucleotide sequences containedwithin said plants or plant cells, and extracts thereof; bacterial orfungal host cells which have been transformed to contain any of thenucleotide sequences of the present invention, including expressioncassettes which are designed for use in plants and which are notintended for expression of a Cry1Bb or a Cry1Bb variant amino acidsequence in said bacterial or fungal host cells, and the like.

The expression cassettes and the coding sequences contained therein andthe proteins expressed therefrom, i.e., the subjects of the presentinvention, can be introduced into a wide variety of microbial or planthosts. In some embodiments of the present invention, transformedmicrobial hosts can be used in preliminary steps for preparingprecursors, for example, that will eventually be used to transform, inpreferred embodiments, plant cells and plants so that the plant andplant cells express the insecticidal Cry1Bb or variant proteins from theexpression cassettes or coding sequences or substantial equivalents ofthe present invention. Bacillus, Salmonella, Clostridia, Escherichia,Yersinia, Pseudomonas, Pasteurella, Aeromonas, Agrobacterium,Rhizobacterium, and the like are representative genus' of bacteriawhich, when transformed with sequences of the present invention, arewithin the scope of the present invention, and methods are well known inthe art for transforming and selecting recombinant microbes within thescope of the present invention.

In preferred embodiments, expression of the proteins of the presentinvention from the non-native nucleotide sequences of the presentinvention and from the expression cassettes of the present invention inplant cells, plant tissues, and plant hosts are within the scope of theinvention. Methods for introducing heterologous nucleotide sequencesinto plant cells, plant genomes, plant chloroplasts and plastids and thelike are well known in the art and include but are not limited toballistic transformation methods, Agrobacterium or Rhizobacteriummediated transformation, vacuum mediated DNA uptake transformationmethods, protoplast fusion methods, and the like are well known in theart and are within the scope of the present invention. These methods canbe used for introducing a nucleotide sequence of the present inventioninto a plant cell, for example, into a crop plant such as corn, wheat,rice, oat, cotton, soybean, sunflower, cauliflower, broccoli, canola orrape seed, and the like. In addition, fruit trees such as apples, pears,peaches, apricot, orange, lemon, lime, grapefruit, and the like, andvines such as grapes, and berries such as blueberries and strawberries,potato, sugar cane, beans and the like, and grasses such as bluegrass,brome, crabgrass, creeping bentgrass, fescue, ryegrass, Saint Augustine,timothy, zoysia, and the like and forage plants such as alfalfa, andclover, and the like, are within the scope of the present invention. Thenucleotide sequences encoding Cry1Bb and amino acid sequence variantsand the expression cassettes of the present invention are particularlywell suited as exemplified herein for providing high-level expression ofthe Cry1Bb insecticidal proteins, insecticidal fragments, andinsecticidal variants thereof in planta.

Agronomically and commercially important products and/or compositions ofmatter including but not limited to animal feed, commodities, and cornproducts and by-products that are intended for use as food for humanconsumption or for use in compositions that are intended for humanconsumption including but not limited to corn flour, corn meal, cornsyrup, corn oil, corn starch, popcorn, corn cakes, cereals containingcorn and corn by-products, and the like, and transgenic Cry1Bb broccoli,transgenic Cry1Bb cauliflower, transgenic Cry1Bb squash, transgenicCry1Bb melons, transgenic Cry1Bb cucurbits, transgenic Cry1Bb soybean,transgenic Cry1Bb canola, transgenic Cry1Bb wheat, transgenic Cry1Bbtomatoes, transgenic Cry1Bb fruit trees, and the like are intended to bewithin the cope of the present invention if these products andcompositions of matter contain detectable amounts of the nucleotidesequences or Cry1Bb proteins set forth herein.

As set forth in the examples below, the inventors herein demonstratethat a synthetic nucleotide sequence encoding an insecticidal variantamino acid sequence substantially equivalent to the native Cry1Bb1insecticidal protein exhibits high levels of expression in plants, inparticular when the nucleotide sequence is embedded within a largernucleotide sequence designed for expression of a coding sequence such asthe synthetic sequence when present in plant cells. Therefore, theexpression cassette, and the nucleotide sequence encoding the Cry1Bbprotein, are excellent insect resistant management tools, in particularwhen combined with other Bt or other types of insect toxin proteinsco-expressed along with the Cry1Bb protein or when combined withtopically applied insecticidal chemical agents, each exerting theirspecific insecticidal activity upon a target insect by means of adifferent mode of action than that exhibited by the Cry1Bb protein.

The inventors herein set forth examples of how these insecticidal agentswork, in particular by using Cry1A type resistant Diamondback Moth andCry1A type resistant European Corn Borer. Larvae exposed to Cry1Aproteins exhibit virtually no level of inhibition. However, exposure ofthese Cry1A resistant larvae to Cry1Bb protein results in mortality,indicating that the Cry1B protein functions to cause insecticidaleffects for these species in a way that is different from the means usedby the Cry1A toxins. The inventors therefore demonstrate the utility ofthe protein as a resistance management tool, and demonstrate theimprovement in levels of expression of the Cry1Bb protein in plants fromthe unique and novel expression cassettes disclosed herein.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 In Vitro Bioactivity of Cry1Bb against Dipel™ ResistantEuropean Corn Borer

Lepidopteran species that develop resistance to insecticidal proteinsderived from Bacillus thuringiensis or Bt) bacteria tend to do sothrough multiple, unexpectedly dominant alleles. The development ofresistance to insecticidal proteins under laboratory conditions appearsto be more complex and more difficult to control than many experts haveassumed and could be of importance to regulatory officials responsiblefor monitoring crops that are engineered to produce such proteins. It ispossible that target plant pests could develop resistance in the wild tobiological pesticidal agents such as B. thuringiensis crystal toxinproteins. An extensive review of the literature in this area can befound in Ferre et al. (Annu. Rev. Entomol. 2002,47:501-533).

Recombinant plants that express Cry1A B. thuringiensis crystal proteintoxins have been commercialized since 1996. Requirements for resistancemanagement strategies have been implemented in order to decrease thelikelihood of the development of resistance. Statistical studiesindicate that pest resistance to the Cry1A class of proteins is likelyto develop without the implementation of resistance managementstrategies, and even then, likely to develop if the Cry1A plants aremaintained in the fields in the absence of an additional insecticidalagent exhibiting a mode of action different from the mode of action ofthe Cry1A protein toxin. It has been demonstrated with chemicalinsecticides and with antibiotic selection that resistance is lesslikely to develop when agents exhibiting different modes of action areused in combination and directed to a common insect pest species. Cry1Aresistant strains of lepidopteran larvae have been developed undertightly controlled laboratory conditions. In particular, a Cry1A-typediamondback moth race has been identified which is insensitive to highlevels of Cry1A toxin. It is logical to assume that a pest sensitive toboth Cry1A and Cry1B type toxins would be insensitive to Cry1B typetoxins if the pest develops resistance to Cry1A type toxins. Thisassumption is based primarily on the degree of relationship of Cry1A toCry1B proteins. These proteins belong to the same Cry1 class of B.thuringiensis δ-endotoxin proteins, and are ontologically related.However, Donovan et al. (WO95/04146) demonstrated that diamondback mothstrains resistant to Cry1A-type B. thuringiensis δ-endotoxins retainsensitivity to Cry1Bb, highlighting the utility of this protein as aresistance management tool. In the absence of resistance managementstrategies employing two or more modes of action, Bt toxin levels incompositions used for on planta (topical) application or for in plantaexpression should be maintained at high levels in order to prevent orsignificantly delay the onset of resistance. Alternatively, combining Bttoxins exhibiting different modes of action, i.e., each toxin beingtoxic to the same insect species but each toxin exerting it's effect bya means different from that of the other toxin, would also be a meansfor preventing the onset of resistance.

Donovan et al. demonstrated bioactivity of Cry1Bb1 in in vitro bioassaysagainst a number of lepidopteran species. In particular, bioactivity wasdemonstrated against gypsy moth (Lymantria dispar), European corn borer(Ostrinia nubilalis), fall army worm (Spodoptera frugiperda), soybeanlooper (Pseudoplusia includeizs), diamondback moth (Plutellaxylostella), and cabbage looper (Trichoplusia ni).

The inventors herein demonstrate that a synthetic sequence encoding aCry1Bb insecticidal protein toxin exhibits high levels of expression inplants, and is therefore an excellent insect resistance management tool,in particular when combined with other Bt or other types of insect toxinproteins or chemical agents, each exerting their specific insecticidalactivity upon a target insect by means of a different mode of actionthan that exhibited by Cry1Bb.

Cry1Bb bioactivity against a variety of lepidopteran insects such asEuropean corn borer (ECB, Ostrinia nubilalis)) and fall army worm (FAW,Spodoptera frugiperda) has previously been demonstrated (Donovan et al.,U.S. Pat. Nos. 5,679,343 & 5,616,319). Diamondback moth strainsresistant to Cry1A-type B. thuringiensis δ-endotoxins retain sensitivityto Cry1Bb, highlighting the utility of this protein as a resistancemanagement tool (Donovan et al., supra). ECB is presently controlled ona significant portion of the planted transgenic maize acreage byexpression of Cry1A-type B. thuringiensis δ-endotoxins. This presents anopportunity for the development of ECB populations resistant toCry1A-type B. thuringiensis δ-endotoxins.

A population of ECB selected in the laboratory for resistance to DIPEL™,a commercially available mixture of Bacillus thuringeisis sporescomprising Cry1A-type and Cry2A-type endotoxins, was tested forsensitivity to Cry1Bb to determine if Cry1Bb could control Cry1A-typeresistant ECB (Huang et al., Science 284:965-967; 1999). The test wasconducted by exposing larvae to solubilized B.t. δ-endotoxinincorporated into an artificial diet. Typical levels of Cry1Ab that areattained in commercially available transgenic maize ranges from about 10to about 20 ppm. The results are shown in Table 1. Cry1Ab resistant ECBwere insensitive to levels of Cry1Ab which have not been attained incommercially available transgenic plants. However, these same Cry1Abresistant ECB retained sensitivity to Cry1Bb at levels routinelyattained in transgenic plants as described herein below. These resultssuggest that Cry1A resistant ECB, and presumably other lepidopteranlarvae, which develop resistance to Cry1A type δ-endotoxins shouldexhibit sensitivity to Cry1Bb. TABLE 1 ECB sensitivity to Cry1Bb Dipel ™Resistant ECB Dipel ™ Sensitive ECB Endotoxin (LC50 in ppm) (LC50 inppm) Cry1Ab >50 ppm 0.08-0.4 ppm Cry1Bb 0.32-1.6 ppm <0.32 ppm

Example 2 Construction of Synthetic Nucleotide Sequences Encoding Cry1Bb

Coding sequences derived from Bacillus thuringiensis do not expresswell, if at all, in plants, in general because plant nucleic acidsequences tend to exhibit from about 50% to about 60% or greater GCcontent, while nucleic acid sequences derived from Bacillusthuringiensis tend to exhibit from about 60 to about 70% AT content.Generally, it has been demonstrated that reduction of AT rich sequencesin BT protein encoding regions intended for expression in plants resultsin improvements in in planta levels of expression of the coding region.One means for decreasing the level of AT composition in Bt codingsequences comprises obtaining the amino acid sequence of a Bt proteinand constructing a gene for expression in plant cells by using wherepossible a codon for each particular amino acid in the protein sequencewhich reduces the overall composition of AT in the coding sequence suchthat the overall GC content of the coding sequence tends to be fromabout 50% to about 60% or greater, and which results in a codingsequence which is substantially devoid of regions containing stretchesof A or T or A and T of less than five or six nucleotides in length.Examples of non-native nucleotide sequences for use in in plantaexpression of Cry1Bb and Cry1Bb amino acid sequence variants, analogs,and homologs are illustrated at SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8,SEQ ID NO:11, and SEQ ID NO:13, the designated Cry1Bb open readingframes of which correspond to amino acid sequences comprising a Cry1Bbinsecticidal protein or insecticidal fragment thereof as set forth inSEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.

The nucleotide composition of each of the coding sequences intended forimproved expression of Cry1Bb toxins or insecticidal fragments thereofas set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, andSEQ ID NO:13 are comprised of between 55 and 65% GC. These non-nativeand synthetic sequences encoding Cry1Bb amino acid sequences and Cry1Bbamino acid sequence variants were constructed according to the method ofBrown et al. substantially as set forth in U.S. Pat. No. 5,689,052,except that the resulting nucleotide sequence was not partially obtainedfrom starting material originating from native B. thuringiensisnucleotide sequences. Instead, the complete synthetic Cry1Bb codingsequence was prepared by nucleotide synthesis service providers afterproviding one or more nucleotide synthesis service providers with all ora part of the desired terminal or resulting nucleotide sequence forencoding Cry1Bb in plants. The resulting sequences comprise pre-selectednucleotide sequences encoding at least an insecticidal portion orfragment of a Cry1Bb, or a Cry1Bb amino acid sequence variant, whereinthe pre-selected nucleotide sequence is adjusted relative to the nativenucleotide sequence to be more efficiently expressed in plants incomparison to the levels of expression of the native nucleotide sequenceencoding a Cry1Bb insecticidal protein. While the nucleotide sequencesdisclosed herein are but a few examples of Cry1Bb coding sequences whichare shown herein to function in plants to produce insect inhibitoryeffective amounts of Cry1Bb in plant cells and in plant tissues, itshould be understood that there are multiples of other sequences whichmay work as well to allow for expression of Cry1Bb in plants, keeping inmind the limitations on codon usage and specific nucleotide compositiondescribed herein above. These sequences can be linked to plantfunctional promoters and 3′ end transcription termination andpolyadenylation sequences, as well as other types of expressionmodulating elements for optimizing the expression of each sequence in adesired genus, species, or variety of plant cell or plant tissue. It isbelieved that a nucleotide sequence encoding all or an insecticidalfragment of a Cry1Bb or a Cry1Bb amino acid sequence variant, or thelike, which is identical to or approximately between 95-99% identical tothe sequences set forth herein would function as well as those sequencesdescribed herein for expression of said protein or proteins in plants,and are specifically intended to be within the scope of the presentinvention.

Example 3 Cassettes Encoding Cry1Bb and Variants for Use in Plants

A variety of genetic elements were combined together with Cry1Bb codingsequences in plant transient expression and transformation vectors inorder to identify sequences comprising plant expression cassettes likelyto provide commercially useful levels of expression of Cry1Bb protein inplants. The individual elements selected for use herein are exemplaryonly, and in the examples herein, the elements selected were chosenparticularly because the exemplary plants tested herein are maize plantsand the selected elements have been previously shown to function inmaize plants as promoters, intronic sequences, plastid targetingsequences, leader sequences, and termination sequences. Variouspromoters, 5′ untranslated leaders, intron sequences, plastid targetingsequences, and 3′ end transcription termination and polyadenylationsequences were grouped together in operable combinations with syntheticCry1Bb coding sequences. Promoters were selected from theCAMV-e35S(P-CaMV.e35S) promoter and the figwort mosaic virus (P-FMV.35S)promoter, however, the skilled artisan will recognize that many otherplant functional promoters known in the art will suffice in place of thetwo selected for exemplary purposes. Other elements are to be construedas being exemplary as well. Untranslated leader sequences were selectedfrom the wheat chlorophyll a/b binding protein leader (L-Ta.Cab), andthe rice beta tubulin leader (L-Os.βTub). Intronic sequences wereselected from the rice actin 1 gene intron (1-Os.Act1) and the ricephenylalanine ammonia lyase gene intron (1-Os.PAL). A nucleotidesequence encoding a Zea mays ribulose bis-phosphate carboxylase smallsubunit plastid targeting sequence was used in some vector constructions(TS-Zm.rbcs) (Lebrun et al., 1987, NAR 15:4360). The nucleotide sequenceencoding the Zea mays plastid targeting peptide is set forth herein atleast from nucleotide position 2644 through nucleotide position 3040 ofSEQ ID NO:8, and consists of a maize genomic coding fragment containingan intron sequence (nucleotide 2791 through nucleotide 2953 of SEQ IDNO:8) as well as a sequence encoding a duplicated proteolytic cleavagesite present in the resulting plastid targeting peptide amino acidsequence (first of said sequences encoding said duplicated cleavagesites being positioned from nucleotide positions 2644 through 2790 andfurther, after excision of the intron, including the nucleotides atposition 2954 through 3040, and the second of said sequences encodingsaid duplicated cleavage sites being positioned within the amino acidsequence encoded by nucleotides 2954 through 3040 of SEQ ID NO:8, andderived from plastid targeting sequence zmS 1; Russell et al., 1993).Direct translational fusions of the TS-Zm.rbcs to the amino terminus ofthe preferred sequences encoding insecticidal proteins herein are usefulin obtaining elevated levels of the insecticidal protein in transgenicmaize.

In-frame fusions of the TS-Zm.rbcs nucleic acid sequence (as set forthat nucleotides 2644 through 3040 of SEQ ID NO:8) to the gene sequenceencoding a Cry1Bb protein (SEQ ID NO:3) can be effected by ligation ofthe NcoI site at the 3′ (C-terminal encoding) end of the TS-Zm.rbcscoding sequence with the 5′ NcoI site (N-terminal encoding) of theCry1Bb coding sequence. The use of plastid targeting sequences linked toa Cry1A or a Cry2Ab insecticidal toxin protein has been demonstrated tobe effective in improving the level of protein accumulation in a plantcell. However, it is not known which Bt proteins can benefit from thefunction of a linked plastid targeting peptide (see Corbin et al., WO00/26371). Transcription termination and polyadenylation sequences wereselected from the wheat Hsp17 gene termination sequence (T-Ta.Hsp17) andthe rice lactate dehydrogenase gene termination sequence (T-Os.Ldh), andare identified as features by sequence location within cassettesequences provided herein.

In order to effectively monitor levels of expression of Cry1Bb intransient expression systems and in transgenic plants, immunologicalassays were developed using antibodies specific for binding to Cry1Bbprotein. Antibodies to purified Cry1Bb protein were produced by meanswell known in the art. Quantitative ELISA assays were developed formeasuring Cry1Bb protein levels in various assays and compositions ofmatter. A Cry1Bb pure protein crystal slurry was obtained from Bacillusthuringiensis strain EG7283 (NRRL B-21111, Donovan et al., U.S. Pat. No.5,679,343). The crystals were solubilized, and the protein quantifiedand sent to a service provider for polyclonal antisera generation(Celsis Laboratory, St. Louis). Rabbits were immunized with the antigenaccording to standard immunization procedures, resulting in a high titerCry1Bb antisera.

IgG was purified from the rabbit sera and used as a capture antibody ina sandwich ELISA. The ELISA assay was performed by first coating a96-well polystyrene ELISA plate (Nunc, Denmark) with a high titerpolyclonal anti-Cry1Bb capture antibody at a concentration of 125 ngIgG/well. The plate was allowed to incubate overnight at 4° C. in asealed, humid container. The following day, the plate was washed andsamples were loaded beside a standard curve comprising purified Cry1Bbprotein. Appropriate buffer blanks and positive/negative controls wereincluded. The Cry1Bb test samples, standards and controls were incubatedovernight at 4° C. with the bound capture antibody and a horseradishperoxidase-conjugated secondary antibody. The following day, plates werewashed and treated with a TMB substrate solution to allow for acolorimetric detection. Concentrations of Cry1Bb were determined in eachsample by extrapolating an optical density reading against a Cry1Bbstandard curve. Results are reported on parts per million, fresh weightbasis.

Four distinct expression cassettes were tested in transient cornprotoplast expression assays and evaluated for expression byquantitative ELISA and efficacy against ECB in diet overlay bioassay.The vectors and elements tested are outlined in Table 2. TABLE 2Composition of Corn Protoplast Cry1Bb Transient Expression Vectors andExpression Cassettes 33731^(a) P-FMV : L-Os.βTub : I-Os.PAL : cry1Bb1 :T-Os.Ldh SEQ ID NO:5 40227^(a) P-CaMV.e35S : L-Ta.Cab : I-Os.Act1 :cry1Bb1 : T-Ta.Hsp17 SEQ ID NO:11 33732^(a) P-FMV : L-Os.βTub : I-Os.PAL: TS-Zm.rbcs : cry1Bb1 : T-Os.Ldh SEQ ID NO:8 40228^(a) P-CaMV.e35S :L-Ta.Cab : I-Os.Act1 : TS-Zm.rbcs : cry1Bb1 : T-Os.Ldh SEQ ID NO:13(:) represents separation of various amorphous nucleotides betweenfunctional genetic elements; P indicates promoter element; L indicatesuntranslated 5 leader sequence; I indicates intron sequence; TSindicates transit peptide (containing an embedded intron in thisexample); T indicates plant functional transcription termination andpolyadenylation sequence; SEQ ID NO: indicates the particular sequencelisting number exemplifying the indicated composition and expressioncassette;(a) designates pMON plasmid number corresponding to the operably linkedgenetic elements on same line. Each expression cassette contains asequence encoding an identical Cry1Bb variant amino acid sequence;pMON33731 expression cassette was transferred into a planttransformation vector to create pMON33733. PMON33732 expression cassettewas transferred into a plant transformation vector to create pMON33734.

Expression from the indicated vectors and insecticidal bioactivity ofthe transient protoplasts was tested in a maize transient expressionassay. Cry1Bb protein expression was measured by ELISA as describedabove, and insecticidal activity was measured by feeding transient maizeprotoplasts to ECB larvae. The results obtained are shown in Table 3.TABLE 3 Cry1Bb corn protoplast expression and efficacy against ECBlarvae. Vector ELISA pMON: (ppm) Mortality 33731 0.21 0.92 40227 0.340.92 33732 0.05 0.5 40228 0.1 0.83 no DNA 0 0.17

Vectors encoding Cry1Bb protein not targeted for chloroplast uptakeexpressed greater levels of Cry1Bb protein than vectors encodingplastid-targeted Cry1Bb fusion proteins. However, Cry1Bb proteinexpressed from either form of expression cassette resulted in effectivelevels of mortality in comparison to the negative control, butnon-targeted expression was better likely due to the elevated levels ofCry1Bb protein accumulation. In any event, it is nonetheless clear thateither form of expression cassette would be equally efficacious indelivering Cry1Bb-mediated insect control in transgenic plants.

Example 4 Plant Transformation and Expression

Transgenic corn plants expressing Cry1Bb protein were produced aftertransformation with plant transformation vectors containingsubstantially the same expression cassettes exemplified in the plasmidsas set forth in Table 2. Expression of the Cry1Bb protein produced inthese transgenic corn plant events was compared and was observed to besignificantly higher in plants produced after transformation withvectors containing expression cassettes in which the Cry1Bb protein orvariant was targeted to the chloroplast. pMON33733 contains anexpression cassette as set forth in SEQ ID NO:5 comprising a sequencecontaining an FMV35S promoter (P-FMV), a rice beta tubulin untranslatedleader sequence (L-Os.ptub), a rice phenylalanine ammonia lyase intronsequence (1-Os.PAL), a synthetic Cry1Bb variant coding sequence(cry1Bbl), and a rice lactate dehydrogenase transcription terminationand polyadenylation sequence (T-Os.Ldh). pMON33734 contains anexpression cassette as set forth in SEQ ID NO:8 consisting of a sequencecontaining a FMV35S promoter (P-FMV), a rice beta tubulin untranslatedleader sequence (L-Os.ptub), a rice phenylalanine ammonia lyase intronsequence (I-Os.PAL), a sequence encoding a maize ribulose bis-phosphatecarboxylase small subunit chloroplast transit peptide (CTP orTP-Zm.rbcs) fused in-frame to a synthetic Cry1Bb variant coding sequence(cry1Bb1 variant), and a rice lactate dehydrogenase transcriptiontermination and polyadenylation sequence (T-Os.Ldh). Both vectors alsocontain a cassette consisting of a CaMV35S promoter sequence, a neomycinphosphotransferase (NPI) coding sequence, and a nopaline synthasetranscription termination and polyadenylation sequence that confersparomomycin resistance to transformed plant tissue and is used as aselectable marker. One skilled in the art will recognize that anyelement that can be used as a selectable marker can function in place ofthe present nptII gene. For example luc, bar, phnO, glyphosate tolerantepsps alleles, gox, and the like, can be used along with or in place ofnptII as a selectable marker for identifying plant cells and plants thathave been transformed to contain a gene of interest such as a syntheticsequence encoding an insecticidal protein. Transgenic corn plantsresistant to paromomycin were derived essentially as described in U.S.Pat. No. 5,424,412. Leaf discs from Ro plants were placed in wells withECB larvae and scored for ECB resistance to identify plants expressingtoxic or insect inhibitory levels of Cry1Bb protein. Ninety-six (96)independent events were obtained after transformation with pMON33733 andselection in the presence of paromomycin. Twelve (12) of these wereidentified by leaf disc feeding bioassay to exhibit resistance toEuropean corn borer, and six (6) of these ECB resistant plants exhibitedstrong s resistance. Ninety-four (94) independent events were obtainedafter transformation with pMON33734 and selection in the presence ofparomomycin. Plants in this group exhibited from about one (1) ppm toabout one-hundred sixty (160) ppm of Cry1Bb protein as measured byELISA. Eighteen (18) of these were identified by leaf disc feedingbioassay to exhibit resistance to ECB, and eleven (11) of theseexhibited strong resistance. Plants in this group exhibited from aboutone (1) ppm to about three-hundred forty five (345) ppm of Cry1Bbprotein as measured by ELISA.

Leaf tissue from ECB resistant, independently transformed transgenicevents in the R₀ stage was subjected to quantitative analysis of Cry1Bbprotein levels by the quantitative ELISA assay. Tissue samples fromfresh R₀ corn leaf discs were sampled from each plant directly into a1.5 mL Sarstedt microcentrifuge tube. Plants were sampled at about theV3 leaf stage. Each leaf sample was weighed and TBA buffer (100 mMTrizma Base, pH 7.5; 100 mM sodium borate; 0.2% (w/v) L-ascorbic acid(added immediately before use); 0.05% Tween-20; 5 mM MgCl₂ (6H₂O)) wasadded at a 1:100 tissue to buffer ratio. The leaf tissue was homogenizedinto the buffer with a Wheaton overhead stirrer for ˜20 seconds. Thehomogenized leaf tissue was then subjected to about 12,000 g for 5minutes in a microcentrifuge, separating the plant tissue solids fromthe solubilized protein supernatant. This extract supernatant was addedto wells in microtiter plates and subjected to analysis by D ELISA.

Protein blot analysis confirmed that the increased level ofcross-reactive material produced by pMON33734 events was due toincreased accumulation of an approximately 66 kDa protein thatco-migrates with a 66 kDa protein which accumulates in pMON33733 eventsand which is immuno-reactive with anti-Cry1Bb antiserum. The 66 kDaprotein is consistent in mass with the predicted size of the Cry1Bbtoxin domain and may be derived by proteolysis of the about 130,000 kDafull length Cry1Bb variant protein protoxin after expression in planta.The native Cry1Bb full length protein produced from Bacillusthuringiensis strain EG5847 can be proteolytically cleaved to release aninsecticidal protein which is approximately 66 kDa, corresponding to thecore toxin domain of Cry1Bb, which likely is represented by the aminoacid sequence from about position one (1) through about positionsix-hundred forty three (643) as set forth in SEQ ID NO:2. The datareported herein suggests that the targeting peptide fused to theN-terminus of the Cry1Bb protein and expressed in events transformedwith pMON33734 was efficiently processed or removed, and therefore thatthe insecticidal protein toxin must be localized within the chloroplast.

To establish that events produced from transformation with the plastidtargeted Cry1Bb expression vector pMON33734 resulted in localization ofthe toxin protein to the chloroplast, samples of these plants weresubjected to protein immuno-gold labeling and electron microscopy andcompared to samples from events transformed with the expression vectorpMON33733. Immuno-gold labeling showed the presence of gold particlesand thus Cry1Bb protein only in the chloroplasts within the cellsderived from events produced by transformation with pMON33734,indicating that the protein was properly targeted using the CTPsequence. In contrast, Cry1Bb protein was found throughout the cellsderived from events produced by transformation with pMON33733. Goldlabeling of cells in an isogenic control line, H99, was not apparent.

Events derived from transformation with the pMON33734 vector produced ahigher percentage of events exhibiting ECB tolerance. Leaf disks from Roplants were exposed to neonate ECB larvae and scored for feeding damageas previously described (Armstrong et al, 1995, Crop Science35:550-557). While non-transgenic control disks were totally consumed,disks from transgenic lines exhibiting resistance to ECB feeding werereadily identified. The percentage of events-exhibiting any ECBresistance was markedly increased in events transformed with the vectorpMON33734 (Table 4). Twice as many events with strong ECB resistancewere obtained when pMON33734 was used relative to events selected aftertransformation with the vector pMON33733. Thus, transformation of plantcells using the vector encoding the chloroplast targeted Cry1Bbsurprisingly increases the probability of obtaining a transgenic lineexhibiting insecticidal properties, insect toxicity, and ECB resistance.TABLE 4 Expression of Cry1Bb in R₀ maize Total Total Total Strong 0-1010-50 50-150 150-200 >200 Highest Vector Events¹ ECB R² ECB R³ ppm⁴ ppmppm ppm ppm ppm pMON33733 96 12 6 3 6 2 1 0 160 (non- (12.5%) (6.3%)targeted) pMON33734 94 18 11 5 3 6 2 2 345 (plastid (19%) (12%)targeted)¹Number of paromomycin resistant plant events obtained²Number and percentage of the total (in parenthesis) plants exhibitingECB resistance³Number and percentage of the total (in parenthesis) plants exhibitingstrong ECB resistance.⁴parts per million (or ug/gm fresh weight tissue) of Cry1Bb asdetermined by ELISA.

Example 5 Herbicide Resistant Transgenic Maize Expressing Cry1Bb

The expression cassette in pMON33732, identical to the expressioncassette in pMON33734, as set forth in SEQ ID NO:8, demonstrated insectinhibitory effective levels of Cry1Bb expression in transgenic maize.This expression cassette was subsequently engineered into twoalternative monocotyledonous plant transformation vectors that containan identical gene expression cassette permitting recovery of transgenicmaize plants with glyphosate tolerance. The gene expression cassetteconferring glyphosate tolerance consists of a previously described riceactin Act1 promoter and intron sequence, an Arabidopsis thaliana EPSPSuntranslated leader sequence, a sequence encoding an Arabidopsisthaliana plastid targeting peptide, a sequence encoding a glyphosateinsensitive EPSPS (enol pyruvyl shikimate 3 phosphate synthase) or AroAprotein referred to herein and in the literature as CP4, and a NOS 3′transcription termination and polyadenylation sequence. pMON33750 is acomposite vector containing two expression cassettes. The cassetteexpressing Cry1Bb is identical to the cassette present in pMON33734. Theother cassette encodes a EPSPS enzyme which confers tolerance toglyphosate herbicide as the selectable marker in place of the NptIIcoding sequence in pMON33734. pMON33750 was digested with MluIrestriction endonuclease to release a DNA fragment containing only theCry1Bb and glyphosate tolerance expression cassettes, which was purifiedand used to transform maize cells using ballistic methods, followed byglyphosate selection, using methods well known in the art. Anothercomposite vector containing both the Cry1Bb and glyphosate tolerancecassettes, pMON40213, was used to transform maize cells usingAgrobacterium-mediated transformation, by methods well known in the art.Maize cells transformed with DNA from pMON33750 or with pMON40213 weresubsequently regenerated into glyphosate tolerant plants and screenedfor expression of Cry1Bb protein using the ECB leaf disk feedingbioassay and Cry1Bb quantitative ELISA (Armstrong et al., supra.).

Transgenic pMON33750 and pMON40213 S2 (homozygous, self pollinated)progeny maize plants were subsequently assayed for expression of Cry1Bbprotein. Expression of Cry1Bb protein was detectable at all stages ofdevelopment assayed, with the highest levels detected at the V12 stageof development. This data confirmed that the pMON33750 and pMON40213transgenes remain active after multiple generations and throughout plantdevelopment, two critical characteristics for agronomically usefultransgene-mediated insect control (Table 5). High level insecticidaltransgene expression at later stages of plant development is especiallyuseful in providing season long control of insect pests. TABLE 5Expression of Cry1Bb in Maize at V4, V8 and V12 leaf stages V4² V8² V12²Event¹ (Cry1Bb, ppm) (Cry1Bb, ppm) (Cry1Bb, ppm) pMON33750 1 RAB138 5 326 2 RAB150 7 11 45 3 RAB152 7 8 46 4 RAB158 5 9 36 5 RAB167 10 9 54 6RAB169 11 8 56 7 RAB175 18 9 38 8 RAB183 15 9 64 9 RAB174 16 8 20 10RAB180 12 9 22 11 RAB188 10 14 56 12 RAB201 13 15 44 13 RAB210 12 9 5214 RAB226 11 11 55 15 RAB249 10 9 43 16 RAB252 12 16 72 pMON40213 1RAA376 8 9 55 2 RAA401 5 9 49 LH198 0 0 01-individual events in this column were selected after transformationwith nucleotide sequences present in the 5 plasmid indicated in boldfacetype2-events were sampled at either the 4, 8, or 12 leaf stage and the levelof Cry1Bb protein was determined using ELISA as described herein, andreported as parts per million of total protein

In order to compare levels of ECB control by Bt insecticidal transgenicmaize, three pMON22750 transgenic maize events were grown in fieldconditions and compared to a commercially available transgenic maizeline, MON810 (Monsanto Company, St. Louis, Missouri) expressing a Cry1AB. thuringiensis insecticidal crystal protein toxin. First and secondgeneration European Corn Borer broods (ECB 1 and ECB2, respectively)were evaluated and the results are shown in Table 6. In this experiment,the non-transgenic control sustained extensive damage while thetransgenic maize expressing either a plastid targeted Cry1Bb (RAB172,401, and 150) or Cry1A (MON810) both displayed excellent control of ECB1and ECB2. Control of ECB infestation and feeding damage by plantsexpressing Cry1Bb protein was statistically indistinguishable fromcontrol of ECB infestation and deeding damage by plants expressing Cry1Aprotein.

The stand-alone ECB control exhibited by maize expressing Cry1Bb thussatisfies the key redundant control requirement for an insect resistancemanagement strategy that would be based on a two gene product. This dataand aforementioned diet bioassay data demonstrating activity of Cry1Bbagainst insects that are resistant to Cry1A-type B. thuringiensisδ-endotoxins indicates that maize expressing the Cry1Bb insecticidalprotein could be used to combat infestations of Cry1A-type resistantEuropean corn borer populations. Infestations of Cry1A-type resistantinsects could be controlled either by exclusive use of plants expressingCry1Bb or by genetically combining the Cry1Bb transgene with at leastone additional insecticidal transgene in a single plant (Corbin et al.,WO00/26371). Examples of the second transgene include cry1Aa, cry1Ab,cry1Ac, cry1F, cry2Ab, and various hybrid genes formed from cry1A andcry1F coding sequences expressing chimeras exhibiting the same orimproved insecticidal bioactivity of the native proteins from which thehybrids were formed. All transgenic events expressing an insecticidalCry1 protein exhibited significantly better insect resistance than thecontrol (p<0.05). TABLE 6 Performance of Transgeuic Maize in fieldconditions. Cry ECB1^(A) ECB2 Gene Event 0-9 leaf SE^(B) cm tunnelSE^(C) 1Bb RAB172 0.55 0.63 0.43 1.01 1Bb RAB401 0.20 0.52 0.00 0.83 1BbRAB150 0.07 0.52 0.14 0.83 1A MON810 0.25 0.45 0.32 0.72 Controlnon-transgenic 8.90 0.45 25.08 0.72^(A):leaf damage rating scale of 0-9 where 0 represents no damage/excellent control and a 9 represents extreme damage/ no control.^(B):SE indicates standard error or standard deviation from theindicated leaf damage rating^(C):SE indicates the standard error or standard deviation from theindicated tunneling distance in centimeters

Example 6 Maize Expressing Cry1Bb Exhibits Improved Fall Army WormControl

Although ECB is the primary maize insect pest in North America, otherinsects such as the fall armyworm (FAW or Spodoptera frugiperda) canalso cause significant economic loss, particularly in South America.pMON33750 transformed maize events were challenged with FAW larvae todetermine if transgenic maize expressing Cry1Bb could provide improvedcontrol of insects other than ECB. The results are shown in Table 7.Several events expressing Cry1Bb demonstrated excellent protectionagainst heavy natural FAW infestation in field tests. In at least oneevent (RAB172), FAW control was statistically indistinguishable fromcontrol conferred by plants expressing only Cry2Ab targeted to thechloroplasts or a combination of Cry1A and Cry2Ab. All events exhibitedsignificantly better fall armyworm control than the control plants(p≦0.05). TABLE 7 Leaf Damage Rating of Transgenic Maize ExpressingCry1Bb Infested with Fall Armyworm. FAW^(A) Gene Event 0-9 leaf SE⁸ 1BbRAB172 0.33 0.38 1Bb RAB401 1.78 0.38 1Bb RAB150 0.75 0.38 2Ab MON8400.03 0.38 1A/2Ab MON810/840 0.00 0.38 Control B73/H99 3.33 0.38A:leaf damage rating scale of 0-9 where 0 represents no damage/excellent control and a 9 represents extreme damage no control.B:SE indicates standard error or standard deviation from the indicatedleaf damage rating

Example 7 Lepidopteran Pest Control by Plants Expressing Cry1Bb

Leaf disks from V4 stage transgenic maize plants were exposed to cornearworm (CEW), fall armyworm (FAW), black cutworm (BCW), and Europeancorn borer (ECB) under controlled conditions to determine the effect ofin planta expression of insecticidal amounts of a variant Cry1Bbinsecticidal amino acid sequence. Expression levels of Cry1Bb proteinwas determined from disks derived from the same leaves used for thebioassay. Eight sibling plants per event were evaluated for insecticidalactivity as measured using the leaf damage rating (LDR) scale of 0-11 (0is complete control; 11 is no control, with intermediated levels definedas excellent, good, and marginal). Plants expressing Cry1Bb exhibitedexcellent control of ECB, good control of FAW, marginal control of CEW,and no control of BCW (Table 8). Some control of CEW was also observedwith leaf disks from plants transformed with pMON33750, an unexpectedresult in view of previous diet incorporation assays where CEW waschallenged with solubilized Cry1Bb derived from Bacillus thuringiensis.Leaf disks derived from the commercial event expressing Cry1A, MON810,were used as the positive control and displayed excellent control ofboth ECB and CEW, but no control of FAW, which highlights the utility ofthe Cry1Bb transgene in FAW control. Maize event MON840 expressing agene encoding a chloroplast targeted Cry2Ab insecticidal crystal proteinwas a positive check for control of each of the target pests in thisstudy. TABLE 8 Bioactivity of Cry1Bb Transgenic Maize Against CEW, FAW,BCW, and ECB R1 generation Cry1Bb transgenic plants leaf disk bioassaystudy. CEW FAW BCW ECB Expression Plant Event LDR (0-11) LDR (0-11) LDR(0-11) LDR (0-11) “cry1Bb, ppm” RR99MJV03:438:1 RAB114 4 2 8 1 5.64RR99MJV03:438:2 RAB114 4 1 5 0 4.43 RR99MJV03:438:3 RAB114 5 4 11 1 5.19RR99MJV03:438:4 RAB114 7 1 7 0 6.73 RR99MJV03:438:5 RAB114 6 4 11 0 4.42RR99MJV03:438:6 RAB114 6 3 11 0 3.05 RR99MJV03:438:7 RAB114 4 1 11 03.41 RR99MJV03:438:8 RAB114 8 5 11 1 1.19 RR99MJV03:441:1 RAB138 6 11 110 1.45 RR99MJV03:441:2 RAB138 4 1 11 0 1.61 RR99MJV03:441:3 RAB138 8 411 0 2.86 RR99MJV03:441:4 RAB138 11 2 11 0 2.75 RR99MJV03:441:5 RAB13811 3 11 0 2.87 RR99MJV03:441:6 RAB138 4 1 11 0 1.48 RR99MJV03:441:7RAB138 4 1 11 0 1.45 RR99MJV03:441:8 RAB138 11 4 11 1 1.59RR99MJV03:473:1 RAB169 11 2 11 0 5.39 RR99MJV03:473:2 RAB169 6 1 9 04.96 RR99MJV03:473:3 RAB169 5 3 8 0 5.09 RR99MJV03:473:4 RAB169 7 3 8 13.62 RR99MJV03:473:5 RAB169 5 1 7 1 7.15 RR99MJV03:473:6 RAB169 11 1 110 3.89 RR99MJV03:473:7 RA8169 10 4 11 0 6.08 RR99MJV03:473:8 RAB169 3 18 1 12.74 RR99MJV03:477:1 RAB174 11 5 11 0 6.35 RR99MJV03:477:2 RAB17411 3 11 0 4.19 RR99MJV03:477:3 RAB174 11 2 11 1 6.93 RR99MJV03:477:4RAB174 7 4 11 0 5.57 RR99MJV03:477:5 RAB174 11 2 11 0 3.92RR99MJV03:477:6 RAB174 8 1 11 0 6.31 RR99MJV03:477:7 RAB174 4 3 11 04.25 RR99MJV03:477:8 RAB174 10 1 11 0 3.66 RR99MJV03:483:1 RAB180 4 2 110 8.58 RR99MJV03:483:2 RAB180 2 2 7 0 6.94 RR99MJV03:483:3 RAB180 3 3 110 5.35 RR99MJV03:483:4 RAB180 11 5 11 0 5.02 RR99MJV03:483:5 RAB180 4 17 0 13.68 RR99MJV03:483:6 RAB180 11 2 8 0 9.67 RR99MJV03:483:7 RAB180 44 11 0 4.22 RR99MJV03:483:8 RAB180 4 0 11 0 3.81 RR99MJV03:490:1 RAB1864 1 6 0 8.32 RR99MJVQ3:490:2 RAB186 11 1 11 0 8.59 RR99MJV03:490:3RAB186 11 8 11 0 6.79 RR99MJV03:490:4 RAB186 11 0 11 0 4.8RR99MJV03:490:5 RAB186 6 2 11 0 8.05 RR99MJV03:490:6 RAB186 8 4 6 0 13RR99MJV03:490:7 RAB186 11 1 9 0 4.12 RR99MJV03:490:8 RAB186 5 0 10 03.51 RR99MJV03:492:1 RAB187 8 1 6 0 5.88 RR99MJV03:492:2 RAB187 10 1 9 09.26 RR99MJV03:492:3 RAB187 4 1 6 0 4.76 RR99MJV03:492:4 RAB187 3 1 8 03.84 RR99MJV03:492:5 RAB187 5 2 7 0 4.7 RR99MJV03:492:6 RAB187 8 1 8 04.42 RR99MJV03:492:7 RAB187 11 2 5 0 4.71 RR99MJV03:492:8 RAB187 3 9 6 03.28 RR99MJV03:499:1 RAB196 2 1 11 0 5.76 RR99MJV03:499:2 RAB196 7 2 110 6.73 RR99MJV03:499:3 RAB196 4 7 11 2 5.07 RR99MJV03:499:4 RAB196 8 311 0 5.13 RR99MJV03:499:5 RAB196 11 3 11 0 4.62 RR99MJV03:499:6 RAB196 31 11 0 5.11 RR99MJV03:499:7 RAB196 8 2 11 1 4.38 RR99MJV03:499:8 RAB1969 1 11 0 3.09 RR99MJV03:500:1 RAB196 11 11 2 0 4.25 RR99MJV03:500:3RAB196 7 1 11 0 4.86 RR99MJV03:500:4 RAB196 6 2 5 0 2.95 LH198 (row9) —11 11 6 11 neg. control A1 (row 10) — 11 9 11 11 neg. control ControlMon810 1 11 11 1 cry1Ab Control Mon840 0 0 0 0 cry2Ab

Example 8 Cry1Bb Transgenic Plants Display Improved Insect ResistanceManagement Characteristics under Laboratory and Field Conditions

A plant transformation vector containing a Cry1Bb coding sequence as setforth in SEQ ID NO:3 operably linked upstream to a CaMV35S promoter(P-e35S) and a wheat chlorophyll ab binding protein untranslated leadersequence (L-TaCAB) and downstream to a nopaline synthase 3′ endtranscription termination and polyadenylation sequence (T-AGRtu.nos) wasused to produce Brassica sp. transformation events expressing Cry1Bbamino acid sequence variant insecticidal protein. These plants wereassayed for the ability to control Cry1A-type resistant Diamondback moth(DBM) infestation. Transgenic Brassica sp (Broccoli and Cauliflower) wasobtained by Agrobacterium mediated transformation of cotyledonarypetioles and selection on media containing kanamycin. Transgenic eventsexpressing Cry1Bb were identified by ELISA analysis. Brassica sp.transgenic events were also produced by Agrobacterium mediatedtransformation methods using a kanamycin selectable plant transformationvector which contained an expression cassette comprising a syntheticsequence encoding a Cry1Ac insecticidal protein operably linked upstreamto a CamV35S promoter sequence (P-CaMV35S) and a petunia species Hsp70untranslated leader sequence (L-Pet.Hsp70) and a 3′ end plant functionaltranscription termination and polyadenylation sequence.

Cry1Bb transgenic Brassica sp. plants were challenged in controlledlaboratory conditions where insect mortality could be accuratelymonitored. Broccoli plants expressing Cry1Ac were used as controls andwere infested in parallel with the transgenic plants expressing Cry1Bb.Plants were challenged with cabbage looper, diamondback moth (DBM),Cry1C-resistant diamondback moth (1CrDBM), and Cry1A resistantdiamondback moth (both plant varieties displayed excellent insecticidalbioactivity against cabbage looper, diamondback moth (DBM), andCry1C-resistant diamondback moth (1ArDBM) (Table 9). Three replicateswere used per treatment, and there were twenty (20) larvae per replicateto each plant event. Infestation temperature was maintained at 27 Cthroughout each treatment, and the results were determined atseventy-two (72) hours after infestation. Only the plants expressingCry1Bb exhibited insecticidal activity against the 1ArDBM. Transgeniccauliflower expressing Cry1Bb also displayed excellent control of allspecies tested. Cabbage Looper was also controlled in Cry1Bb cauliflowerevents #2 and #3. TABLE 9 Insecticidal Bioactivity of TransgenicBrassica Plants Expressing Cry 1Ac or Cry1Bb Cabbage Event DBM 1ArDBM1CrDBM Looper % mortality (SEM) Broccoli Cry1Ac #1 100 (0)^(a) 5.00(2.87)^(b) 100 (0)^(a) 100 (0)^(a) Cry1Ac #2 100 (0)^(a)  6.67(1.67)^(b)  96.7 (1.67)^(a) 100 (0)^(a) Cry1Bb #1 100 (0)^(a) 100(0)^(a) 100 (0)^(a) 100 (0)^(a) Cry1Bb #2 100 (0)^(a) 100 (0)^(a) 100(0)^(a) 100 (0)^(a) Cry1Bb #3 100 (0)^(a) 100 (0)^(a) 100 (0)^(a)  61.7(21)^(b) Cry1Bb #4 100 (0)^(a) 100 (0)^(a) 100 (0)^(a)  80 (2.9)^(ab)non-transgenic  0 (0)^(a)  3.33 (1.67)^(b)  5.0 (0)^(b)  3.3 (1.67)^(c)control Cauliflower Cry1Bb #1 100 (0)^(a)  88 (12)^(b) 72 (15)^(c)  15(5.0)^(efg) Cry1Bb #2 100 (0)^(a) 100 (0)^(a) 92 (8.3)^(ab)  93(6.7)^(ab) Cry1Bb #3 100 (0)^(a) 100 (0)^(a) 97 (3.3)^(a) 100 (0)^(a)Cry1Bb#4 100 (0)^(a)  92 (8.3)^(ab) 73 (16)^(bc)  47 (21)^(cde) Cry1Bb#5 100 (0)^(a) 100 (0)^(a) 93 (3.3)^(a)  43 (28)^(cde) non-transgenic 1.7 (1.7)^(b)  0 (0)^(c)  3.3 (3.3)^(d)  1.67 (1.67)^(g) controlvalues in a column followed by the same superscript letter are notsignificantly different from the other values in the column (P < 0.05,LSD); Numbers in parenthesis indicate the extent of variation of theresults in that particular replicate.

Transgenic Brassica sp. were also tested under field conditions forresistance to endemic Lepidopteran insect pest infestations. Typicalinsect infestations in the test location near Weslaco, Texas wereinitiated in the fall season and included cabbage looper, DBM, beetarmyworm, and the great southern white butterfly. Plants were seeded inSeptember and evaluated in December. Plants were evaluated for thenumbers of insect larvae per plant and for the extent of insect feedingdamage. Damage was assessed on ten plants per transgenic event based onthe following zero (0) to five (5) scale: 0—no damage, 1—minor feedingdamage (1% consumed by infesting larvae), 2—minor to moderate damage(2-5% consumed by infesting larvae), 3—moderate damage (6-10% consumedby infesting larvae), 4—moderate to heavy damage (11-30% consumed byinfesting larvae) and 5—heavy damage (>30% consumed by infestinglarvae). The results are shown in Table 10. The data demonstrate thatboth transgenic broccoli and cauliflower transformed to express Cry1Bbor amino acid sequence variants exhibit statistically significantreductions in the number of lepidopteran pest larvae per plant and inthe level of insect damage endured over the course of the growingseason. In broccoli, field performance of plants expressing thetransgene encoding a Cry1Bb protein was indistinguishable from fieldperformance of plants expressing the transgene encoding a Cry1Acprotein. TABLE 10 Field Tests of Lepidopteran Insect Pest Infestation onTransgenic Brassica Plants Expressing Cry1Ac or Cry1Bb mean larvae/plant(N) Mean Damage (N) Broccoli Cry1Ac #1 NT NT Cry1Ac #2 0.63 (19)^(a)0.46 (3)^(a) Cry1Bb #1 0.21 (24)^(a) 0.48 (3)^(a) Cry1Bb #2 NT NT Cry1Bb#3 NT NT Cry1Bb #4 0.13 (15)^(a) 0.43 (3)^(a) 987146-004 14 (19)^(b) 1.9(3)^(b) (neg. Ctrl.) non-transgenic 1.3 (43)^(b) 1.9 (5)^(b) CauliflowerCry1Bb #1 0.12 (25)^(a) 0.0 (3)^(a) Cry1Bb #2 0.21 (19)^(b) 0.07 (3)^(a)Cry1Bb #3 0.00 (4)^(a) 1.25 (1)^(c) Cry1Bb #4 0.31 (26)^(b) 0.52 (3)^(c)Cry1Bb #5 0.29 (17)^(b) 0.30 (3)^(b) non-transgenic 1.6 (41)^(c) 2.1(5)^(d)values in a column followed by the same superscript letter are notsignificantly different from the other values in the column (P<0.05,LSD); Numbers in parenthesis indicate the extent of variation of theresults in that particular replicate.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

All publications and patents mentioned in this specification are hereinincorporated by reference as if each individual publication or patentwas specially and individually stated herein to be incorporated byreference.

1. A polynucleotide sequence optimized for expression of an insecticidalprotein in a plant wherein said polynucleotide sequence comprises asequence selected from the group consisting of from about nucleotideposition 7 through about nucleotide position 1803 as set forth in SEQ IDNO:3, from about nucleotide position 2650 through about nucleotideposition 4446 as set forth in SEQ ID NO:5, from about nucleotideposition 3047 through about nucleotide position 4844 as set forth in SEQID NO:8, from about nucleotide position 1247 through about nucleotideposition 3043 as set forth in SEQ ID NO:11, and from about nucleotideposition 1658 through about nucleotide position 3454 as set forth in SEQID NO:13.
 2. The polynucleotide sequence according to claim 1 whereinsaid sequence is SEQ ID NO:3 from about nucleotide position 7 throughabout nucleotide position
 1803. 3. The polynucleotide sequence accordingto claim 1 wherein said sequence is SEQ ID NO:5 from about nucleotideposition 2650 through about nucleotide position
 4446. 4. Thepolynucleotide sequence according to claim 1 wherein said sequence isSEQ ID NO:8 from about nucleotide position 3047 through about nucleotideposition
 4844. 5. The polynucleotide sequence according to claim 1wherein said sequence is SEQ ID NO:11 from about nucleotide position1247 through about nucleotide position
 3043. 6. The polynucleotidesequence according to claim 1 wherein said sequence is SEQ ID NO:13 fromabout nucleotide position 1658 through about nucleotide position 3454.7. A polynucleotide sequence encoding an insecticidal protein, saidprotein being selected from the group consisting of SEQ ID NO:2 fromabout amino acid position 2 through about amino acid position 600, SEQID NO:4 from about amino acid position 3 through about amino acidposition 601, SEQ ID NO:7 from about amino acid position 3 through aboutamino acid position 601, SEQ ID NO:10 from about amino acid position 3through about amino acid position 601, SEQ ID NO:12 from about aminoacid position 3 through about amino acid position 601, and SEQ ID NO:14from about amino acid position 3 through about amino acid position 601.8. The polynucleotide sequence of claim 7 wherein said polynucleotidesequence encoding said protein is selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:13.9. A expression cassette comprising the polynucleotide sequencesubstantially as set forth in SEQ ID NO:3 which functions in plants toproduce an insecticidal protein, wherein said expression cassette isselected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, and SEQ ID NO:13.
 10. A plant comprising a polynucleotidesequence optimized for expression of an insecticidal protein in a plantwherein said polynucleotide sequence comprises a sequence selected fromthe group consisting of from about nucleotide position 7 through aboutnucleotide position 1803 as set forth in SEQ ID NO:3, from aboutnucleotide position 2650 through about nucleotide position 4446 as setforth in SEQ ID NO:5, from about nucleotide position 3047 through aboutnucleotide position 4844 as set forth in SEQ ID NO:8, from aboutnucleotide position 1247 through about nucleotide position 3043 as setforth in SEQ ID NO:11, and from about nucleotide position 1658 throughabout nucleotide position 3454 as set forth in SEQ ID NO:13.
 11. A seedor progeny produced from the plant of claim 10, wherein said seed orprogeny comprises said sequence selected from the group consisting offrom about nucleotide position 7 through about nucleotide position 1803as set forth in SEQ ID NO:3, from about nucleotide position 2650 throughabout nucleotide position 4446 as set forth in SEQ ID NO:5, from aboutnucleotide position 3047 through about nucleotide position 4844 as setforth in SEQ ID NO:8, from about nucleotide position 1247 through aboutnucleotide position 3043 as set forth in SEQ ID NO:11, and from aboutnucleotide position 1658 through about nucleotide position 3454 as setforth in SEQ ID NO:13.
 12. A plant cell comprising a polynucleotidesequence optimized for expression of an insecticidal protein in a plantwherein said polynucleotide sequence comprises a sequence selected fromthe group consisting of from about nucleotide position 7 through aboutnucleotide position 1803 as set forth in SEQ ID NO:3, from aboutnucleotide position 2650 through about nucleotide position 4446 as setforth in SEQ ID NO:5, from about nucleotide position 3047 through aboutnucleotide position 4844 as set forth in SEQ ID NO:8, from aboutnucleotide position 1247 through about nucleotide position 3043 as setforth in SEQ ID NO:11, and from about nucleotide position 1658 throughabout nucleotide position 3454 as set forth in SEQ ID NO:13.
 13. Amethod for producing a transgenic plant cell expressing an insecticidalCry1Bb endotoxins fragment, said method comprising transforming a plantcell with a polynucleotide sequence comprising a plant functionalpromoter operably linked to a nucleotide sequence encoding said fragmentwherein said nucleotide sequence is selected from the group consistingof from about nucleotide position 7 through about nucleotide position1803 as set forth in SEQ ID NO:3, from about nucleotide position 2650through about nucleotide position 4446 as set forth in SEQ ID NO:5, fromabout nucleotide position 3047 through about nucleotide position 4844 asset forth in SEQ ID NO:8, from about nucleotide position 1247 throughabout nucleotide position 3043 as set forth in SEQ ID NO:11, and fromabout nucleotide position 1658 through about nucleotide position 3454 asset forth in SEQ ID NO:13.
 14. A method for producing a transgenic plantresistant to lepidopteran insect infestation comprising: a) transforminga plant cell with a polynucleotide sequence comprising a plantfunctional promoter operably linked to a nucleotide sequence encoding aninsecticidal. Cry1Bb delta endotoxin fragment; and b) regenerating atransgenic plant from said plant cell, wherein said transgenic plantcomprises said polynucleotide sequence and expresses insecticidallyeffective amounts of said fragment.
 15. A method for producing atransgenic plant resistant to insect infestation comprising breedingtogether a) a first plant transformed to contain a first nucleotidesequence encoding a first Bt insecticidal protein and a first selectablemarker with b) a second plant transformed to contain a second nucleotidesequence different from the first, wherein said second nucleotidesequence encodes a second Bt insecticidal protein different from thefirst, and a second selectable marker different from the first whereinsaid transgenic plant comprises both the first and the second nucleotidesequences; wherein the first and the second selectable markers areselected from the group consisting of antibiotic resistance genes,herbicide resistance genes, and genes encoding enzymes that react with asubstrate to form a product that is visually or immunologicallyobservable; wherein the first Bt insecticidal protein comprises aninsecticidal fragment of a Cry1Bb protein as set forth in SEQ ID NO:3from about nucleotide position 7 through about nucleotide position 1803;and wherein the second Bt insecticidal protein is selected from thegroup of toxins consisting of a Cry1, Cry2, Cry3, Cry4, Cry5, Cry6,Cry9, Cry22, a Cry binary toxin, a VIP toxin, a TIC901 or related toxin,and combinations thereof.
 16. The method of claim 15 wherein saidherbicide resistance genes are selected from the group consisting of agox gene, a gene encoding an EPSPS that is insensitive to glyphosateinhibition, a phnO gene, a bar gene, and a glyphosate acetylase gene.17. A nucleotide sequence encoding at least an insecticidal fragment ofa Cry1Bb delta endotoxin protein, said protein comprising an amino acidsequence as set forth in SEQ ID NO:4 from about amino acid position 3through about amino acid position 601, wherein said nucleotide sequencehybridizes under stringent conditions with a nucleotide sequence as setforth in SEQ ID NO:3 from about nucleotide position 7 through aboutnucleotide position
 1803. 18. A composition comprising an insecticidallyeffective amount of a Cry1Bb endotoxin protein or insecticidal fragmentthereof expressed in a plant from a segment of a nucleotide sequence asset forth in SEQ ID NO:3 from about nucleotide position 7 through aboutnucleotide position 1803 or from a nucleotide sequence encoding saidprotein or fragment thereof that hybridizes to said segment.
 19. Abiological sample derived from a plant, tissue, or seed, wherein saidsample comprises a nucleotide sequence which is or is complementary to asequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:13, and wherein said sequenceis detectable in said sample using a nucleic acid amplification ornucleic acid hybridization method.
 20. The biological sample of claim 19wherein said sample is selected from the group consisting of corn flour,corn meal, corn syrup, corn oil, corn starch, and cereals manufacturedin whole or in part to contain corn by-products.
 21. An extract derivedfrom a corn plant, tissue, or seed comprising a nucleotide sequencewhich is or is complementary to a nucleotide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,and SEQ ID NO:13.
 22. The extract of claim 21 wherein said sequence isdetectable in said extract using a nucleic acid amplification or nucleicacid hybridization method.